Method of inducing a CTL response

ABSTRACT

Disclosed herein are methods for inducing an immunological CTL response to an antigen by sustained, regular delivery of the antigen to a mammal so that the antigen reaches the lymphatic system. Antigen is delivered at a level sufficient to induce an immunologic CTL response in a mammal and the level of the antigen in the mammal&#39;s lymphatic system is maintained over time sufficient to maintain the immunologic CTL response. Also disclosed is an article of manufacture for delivering an antigen that induces a CTL response in an animal.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.09/776,232, filed Feb. 2, 2001 now U.S. Pat. No. 6,977,074, which is acontinuation-in-part of U.S. patent application Ser. No. 09/380,534,filed Sep. 1, 1999 now U.S. Pat. No. 6,994,851, which is a nationalstage entry of PCT Application No. PCT/US98/14289, filed Jul. 10, 1998,which is a continuation-in-part of U.S. patent application Ser. No.08/988,320, filed Dec. 10, 1997 now abandoned and which claimed priorityto Canadian Patent Application No. 2,209,815, filed Jul. 10, 1997, eachof which is hereby expressly incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to a method of inducing a CTL response to anantigen by sustained, regular delivery of the antigen to an animal sothat the antigen reaches the lymphatic system.

BACKGROUND OF THE INVENTION

Cytotoxic T lymphocytes (CTL) are white blood cells found in the blood,spleen and lymph. CTL have the ability to attack and kill other cells ofthe body in a highly specific manner. When CTL are stimulated byspecific antigen, they migrate through the tissues of the body on a“search and destroy” mission for cells bearing the specific antigen.Whether of viral origin or tumor associated, CTL detect antigen that isbound to major histocompatability complexes (MHC) on the surface ofpotential target cells. Once CTL have identified the antigen on the cellsurface, their function is to deliver a lethal hit to the cell.

Although there are hundreds of millions of CTL that reside in thespleen, each individual CTL exclusively responds to a unique andspecific antigen. These individual CTL, dubbed CTL precursors (CTLp),undergo cell division or proliferate upon activation by specific antigento produce daughter cells with precisely the same antigen specificity asthe parent cell. This proliferation increases the total number, and thusthe frequency, of that specific CTLp in the body. A proportion of thesenewly generated CTL briefly recirculate through the body (termedeffector CTL), and have the ability to identify and destroy cellsbearing the specific antigen which they recognize. A significant body ofexperimental evidence suggests that CTL specific for tumor antigens caninhibit tumor growth. Unfortunately, most tumors have only a very weakcapacity to stimulate CTL responses and there has been no means ofinducing a CTL response then sustaining it over a period of timesufficient to continuously inhibit tumor growth. While many attempts todirectly increase the capacity of tumor cells to stimulatetumor-clearing CTL responses in patients have been made, such attemptshave met with limited success. Technical advances over the past tenyears have, however, enabled the identification of natural peptideantigens that are present on tumor cells and which are recognized byCTL. These antigen targets include proteins expressed in significantoverabundance, abnormally expressed embryonic proteins, protein productsfrom mutated oncogenes or suppressor genes, or proteins derived fromcancer-causing viruses present in tumor cells. The challenge has been tofind a way in which to administer an antigen so that it induces anantitumor CTL response and maintains it over time. While many attemptshave now been made to use these antigens clinically in a vaccine, theresults have been less than satisfactory.

An explanation of why CTL therapies have been largely ineffective ateradicating or controlling tumors in a clinical setting include thefollowing:

-   -   (a) Vaccine designs have been inadequate at initiating strong        CTL responses;    -   (b) Tumor cells can down regulate MHC molecules, resulting in        the loss of antigen presentation from the surface of cells,        thereby escaping detection by CTL;    -   (c) After induction, effector CTL recirculation through the body        is highly transient;    -   (d) After recirculation, CTL return to the spleen where they        reside in a nonactive or resting state, and an increase in the        numbers of CTLp residing in the spleen does not reflect active        CTL immunity;    -   (e) In the case of tumors, regrowth of residual tumor cells        following immunization goes undetected by CTLp residing in        spleen in a “resting” state;    -   (f) Because CTL-stimulating antigen presenting cells (APC are        targeted for destruction by the same CTL that they have        activated, the CTL response is self-limiting, which precludes,        under normal circumstances, the continuous stimulation for a        long-lived CTL response.

A growing repertoire of tumor associated antigens are being discoveredthat are recognized by CTL. A variety of techniques have been suggestedto render these antigens effective in CTL vaccines. These includeimmunization using synthetic peptide antigens mixed with animmunostimulatory adjuvant, such as the bacterial toxin BCG;inununization with multiple antigenic peptide systems (MAPS),immunization with “professional” antigen presenting cells, which areisolated from the patient, pulsed with peptide antigen and inoculatedback into the patient as a vaccine; immunization with peptides designedto stimulate both CTL and T helper cell populations; immunization withviruses or bacteria engineered to express tumor antigens; andimmunization with polynucleotide expression vectors (so called DNAvaccines). Unfortunately, none of these approaches has been anunqualified success, As discussed above, the lack of vigoroustherapeutic effects with these vaccine platforms reflects at least tosome degree problems associated with inducing a strong initial CTLresponse and with maintaining ongoing “active” CTL immunity.

Studies by Glenny during the first quarter of the century revealed thataluminum compounds could enhance the strength of diphtheria vaccines.This was ostensibly the first of a long history of observationssupporting a “depot” theory of immunization, which postulates thatantigen slowly leaking into the tissues over an extended time correlateswith the antigenic potency of a vaccine. Today, this antigen depotparadigm forms the intellectual backdrop to most adjuvant developmentprograms. In one form or another, depot type adjuvants are intended toprolong the course of antigen delivery, by forming a lesion at the siteof injection, or simply by the slow degradability of the adjuvantitself, which mixed with the specific antigen forms a depot at the siteof injection. A second function generally attributed to adjuvants aretheir immunostimulatory effects, which appears to trigger the immunesystem to respond to the vaccine. However, adjuvants are a double-edgedsword. They have inherent toxicities. But it is a feature of thesetoxicities that achieves a desired immunostimulatory and/or depoteffect. Side effects such as tissue damage and granulornatous reactionat the site of injection, fever, and in some cases systemic reactions,such as Reiter's syndrome-like symptoms, uveitis and arthritis, are someof the risks associated with the use of adjuvants. Currently, the onlyadjuvant approved by the FDA is alum. It is relatively safe but doeshave side effects such as erythema, subcutaneous nodules, contacthypersensitivity, and granulornatous inflammation. More importantly,alum only acts to potentiate a limited number of antigens, and it verypredominantly stimulates humoral antibody responses rather than CTLimmunity. Thus so far adjuvants have proved to be very ineffectivecomponents for vaccines aimed at inducing clinically relevant CTLresponses.

Recent attempts to induce CTL responses using dendritic cells or otherantigen presenting cells, despite being cumbersome, have shown somepromise. New recombinant virus or bacterial systems carrying genes forspecific antigen are effective at inducing primary CTL responses. Themost effective viruses, for example, that induce strong CTL responsesare those which replicate aggressively in the host. Yet because of therisk for serious or lethal complications as a result of infection,recombinant virus used in a cancer vaccine must be only weaklyreplicative, or be completely replication deficient. This trade-offbetween virulence and efficacy is at present an intractable problem.

DNA (or polynucleotide) vaccines are also being developed for thepurpose of inducing CTL immunity. Once again, the system has intrinsiclimitations that preclude its efficacy in inducing long-lasting CTLimmunity. The DNA vaccines consist of a plasmid or similar geneticconstruct for expressing the antigen of interest. Uptake of the plasmidsystem by cells of the body results in expression of the antigen andinduction of CTL. However, once cells expressing the construct havesucceeded in inducing CTL, they are themselves targets for eradicationby the CTL. The CTL inducing effect is thus again transient. Moreover,the polynucleotide vaccines have thus far suffered from poor efficiencyin terms of CTL induction.

With difficulties in achieving strong primary and/or persisting CTLresponses, there are a number of clinical trial groups now usingrepeated injections of cancer vaccines. The use of antigenically complexmaterials in the vaccine formulation, such as recombinant virus, or thecosts associated with repetitive treatment using cultured APC will,however, make such an approach difficult. On the one hand, repetitiveimmunization with antigenically complex materials drives the immunesystem to elaborate a humoral antibody, as opposed to a CTL response,while on the other hand, use of a minimal CTL antigen (such as a nonamerpeptide) which does not efficiently drive an antibody response, has alsofailed to induce a CTL response. Attempts to develop adjuvants thatenhance the immunostimulatory aspects of minimal CTL antigens haveresulted in the production of materials (i.e. adjuvants) that alsoinduce a competing humoral immune response, or, which simply offerlittle CTL stimulatory effect.

It has also been suggested that certain controlled release technologyusing microspheres or liposomes with subunit antigens and peptides mightbe effective to enhance immunogenecity. The combination of sustainedrelease and depot effect is suggested to reduce the amount of antigenneeded and eliminate booster shots. However, the preparation of suchcompositions is difficult and unpredictable, and vaccine formulationsbased on this technology have not been translated into effectiveclinical treatments.

As can be seen from the foregoing, there has been little success atdeveloping a CTL vaccine that is both capable of inducing a strong CTLresponse then sustaining that response over time. The development of avaccine with these capabilities is essential before effective anti-tumortherapy based on CTL immunity can be contemplated.

OBJECTS OF THE INVENTION

An object of this invention is to provide a method for inducing orsustaining a specific CTL immununological response in a mammal overtime.

Another object of this invention is to provide a method for treating amammal having a malignant tumor or infectious disease by inducing andsustaining an immunological attack on the malignant tumor or infectiousdisease in the mammal.

It is a further object of this invention to provide an article ofmanufacture useful for inducing and sustaining a specific immunologicalCTL response in a mammal over time.

It is a further object of this invention to provide an article ofmanufacture useful for treating a mammal having a malignant tumor orinfectious disease, which article is designed to induce and maintain animmunological attack on the malignant tumor or infectious disease in themammal.

It is a further object of this invention to provide a portable devicefor sustained delivery of an antigen to a mammal having a malignanttumor or infectious disease, where the antigen stimulates the mammal'simmune system to attack the tumor or infectious disease and the deviceis located outside the mammal.

It is still a further object of this invention to provide an implantabledevice for sustained delivery of an antigen to a mammal having amalignant tumor or infectious disease, where the antigen stimulates themammal's immune system to attack the tumor or infectious disease.

It is a further object of this invention to provide antigen compositionsand containers therefor that are useful in the methods, devices, and/orarticles of manufacture of this invention.

Other objects of this invention may be apparent to those of skill in theart by reading the following specification and claims.

SUMMARY OF THE INVENTION

In one aspect of the invention, a method is provided for inducing animmunological CTL response to an antigen by sustained, regular deliveryof the antigen to a mammal so that the antigen reaches the lymphaticsystem. In particular, the antigen is delivered to the mammal at a levelsufficient to induce an immunologic CTL response in the mammal and thelevel of the antigen in the mammal's lymphatic system is maintained overtime sufficient to maintain the immunologic CTL response. Preferably,the antigen is delivered directly to the mammal's lymphatic system, suchas to the spleen, a lymph node or lymph vessel.

Also provided is a method of treating an animal having a disease, orbeing predisposed to a disease, to which the animal's immune systemmounts a cell-mediated response to a disease-related antigen to attackthe disease. In this aspect of the invention, a disease-matched antigenis delivered to the animal at a level sufficient to induce an increasedCTL-response in the animal which is then maintained in the animal bysustained, regular delivery of the disease-matched antigen to the animalfor a time sufficient to treat the disease. The sustained, regulardelivery of the antigen is done in a manner that maintains the level ofantigen in the animal's lymphatic system. Preferably, the sustained,regular delivery is achieved by pumping a physiologically-acceptable,composition of the antigen from a device held external of or implantedin the animal's body so that the antigen reaches the animal's lymphsystem. Optionally, a cytokine that is capable of enhancing the CTLresponse is delivered and/or maintained along with the antigen. Diseasesaddressed in this manner include cancer and pathogenic diseases.

In a further aspect of the invention, an article of manufacture isprovided for delivering an antigen that induces a CTL response in ananimal. In particular, the article comprises a reservoir of aphysiologically-acceptable, antigen-containing composition that iscapable of inducing a CTL response in an animal; a pump connected to thereservoir to deliver the composition at a defined rate; a transmissionline to discharge the composition from the reservoir; and, optionally, adelivery line connected to the transmission line, which delivery line isof a size suitable for positioning in the animal and for delivery of thecomposition in a manner that reaches the lymphatic system of the animal.

In a further aspect of the invention, a process is provided forpreparing a system useful for inducing a sustained CTL response in ananimal needing such a response, which comprises placing aphysiologically-acceptable, antigen-containing composition in areservoir having a pump for delivering the composition at a defined ratethrough a transmission line to the animal.

Another aspect of the invention is a method of inducing and/orsustaining an immunological CTL response in a mammal by delivering anantigen in the form of a polypeptide directly to the lymphatic system ofthe mammal. The antigen can be delivered at a level sufficient to inducean immunologic CTL response in the mammal and the level of the antigenin the mammal's lymphatic system is preferably maintained over timesufficient to maintain the immunologic CTL response.

The antigen can be an 8-10 amino acid peptide. Further, the peptidesequence can be derived from a tumor-associated antigen. Examples oftumor-associated antigens include MelanA (MART-I), gp100 (Pmel 17),tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58),CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom/Mel-40, PRAME, p53, H-Ras,HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barrvirus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7,TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1,PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4,Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA),CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1,SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associatedprotein), TAAL6, TAG72, TLP, TPS, and the like.

The peptide sequence also can be derived from a microbial antigen.Further, the antigen can be provided as a component of a microorganismor mammalian cell. Examples of microorganisms include a protozoan, abacterium, a virus, and the like; the mammalian cell can be an antigenpresenting cell, such as, for example, a dendritic cell.

The antigen can be a native component of the microorganism or mammaliancell. The microorganism or mammalian cell can include, for example, anexogenous antigen. Also, the microorganism or mammalian cell can includea recombinant nucleic acid encoding or promoting expression of theantigen. The microorganism or mammalian cell can express atumor-associated antigen, or a microbial antigen native to a secondmicrobial species. The antigen can be provided as an 8-10 amino acidpeptide.

The present invention in another aspect includes a method of inducingand/or sustaining an immunological CTL response in a mammal bydelivering an antigen, in the form of a vector that can include anucleic acid encoding the antigen, directly to the lymphatic system ofthe mammal. The antigen can be delivered at a level sufficient to inducean immunologic CTL response in the mammal and the level of the antigenin the mammal's lymphatic system is preferably maintained over timesufficient to maintain the immunologic CTL response.

The vector can be a plasmid and the like. The vector further can includea bacterium and the like. The bacterium, for example, can includeListeria, Shigella, Salmonella, Escherichia, and the like. The vector,for example, can be a virus, such as, for example, pox viruses,adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, andthe like.

The nucleic acid can encode, for example, a tumor-associated antigen.Examples of tumor-associated antigens include MelanA (MART-I), gp100(Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1,GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom/Mel-40, PRAME,p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR,Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigensE6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3,c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F,5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3(CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

The nucleic acid can encode, for example, a microbial antigen. Examplesof microbial antigens include a viral antigen, a bacterial antigen, aprotozoal antigen, and the like. The nucleic acid can encode, forexample, a protein or other polypeptide. The nucleic acid also canencode an 8-10 amino acid peptide.

The nucleic acid can be plasmid DNA in a formulation comprising about1-10% ethyl alcohol, 0-1% benzyl alcohol, 0.25-0.5 mM EDTA and acitrate-phosphate buffer of pH 7.4-7.8, comprising about 3-50 mM citrateand about 90-200 mM phosphate. For example, the formulation can include1% ethyl alcohol, 1% benzyl alcohol, 0.5 mM EDTA and a citrate-phosphatebuffer of pH 7.4 to 7.8 comprising 50 mM citrate and 100 mM phosphate.

The invention in another aspect provides a method of inducing and/orsustaining an immunological CTL response in a mammal by delivering amicroorganism or mammalian cell directly to the lymphatic system of themammal. The microorganism or mammalian cell are preferably delivered ata level sufficient to induce an immunologic CTL response in the mammaland the level of the microorganism or mammalian cell in the mammal'slymphatic system is preferably maintained over time sufficient tomaintain the immunologic CTL response.

A further aspect of the invention is a method of inducing and/orsustaining an immunological CTL response in a mammal by delivering anucleic acid, capable of conferring antigen expression, directly to thelymphatic system of the mammal. The nucleic acid can be delivered at alevel sufficient to induce an immunologic CTL response in the mammal andthe level of the nucleic acid in the mammal's lymphatic system ispreferably maintained over time sufficient to maintain the immunologicCTL response.

A further aspect of the invention is a method of inducing and/orsustaining an immunological CTL response in a mammal by delivering anon-peptide antigen directly to the lymphatic system of the mammal. Theantigen is preferably delivered at a level sufficient to induce animmunologic CTL response in the mammal and the level of the antigen inthe mammal's lymphatic system is preferably maintained over timesufficient to maintain the immunologic CTL response.

The invention also provides an article of manufacture for delivering anantigen that induces a CTL response in an animal. In particular, thearticle can be an external device. The article can include a reservoirof a physiologically-acceptable, antigen-containing composition that canbe capable of inducing a CTL response in an animal, a pump connected tothe reservoir to deliver the composition at a defined rate, atransmission line to discharge the composition from the reservoir; and,a delivery line connected to the transmission line. The delivery linecan include a catheter of at least 20 mm for positioning in the animaland for delivery of the composition to the lymphatic system of theanimal.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is a graph showing the lysis of target cells by CTL versus theeffector/target ratio when antigen is delivered as a single dose(circles) and when antigen is delivered by a continuous pump(triangles).

FIG. 2 (A and B) are graphs showing the lysis of target cells by CTLversus the effector/target ratio when antigen is delivered as a singledose (circles), when antigen is delivered by a continuous pump(triangles) and negative control (squares) at (A) 36 hours and (B) 7days.

FIG. 2C is a graph showing the footpad swelling versus time when antigenis delivered as a single dose (circles) and when antigen is delivered bya continuous pump (triangles).

FIG. 3 is a graph showing the lysis of target cells by CTL versus thedose of the peptide antigen when the antigen is deliveredsubcutaneously, intravenously and intrasplenically.

FIG. 4 is a bar graph showing tritiated thymidine uptake in CTL cellsinduced by antigen introduced intravenously, intrasplenically andsubcutaneously.

FIG. 5 is a rough schematic of a human lymphatic system.

FIG. 6. Comparison of anti-peptide CTL responses following immunizationwith various doses of DNA by different routes of injection.

FIG. 7. Comparison of anamnestic antiviral CTL responses followingimmunization with various doses of DNA by different routes of injection.

FIG. 8. Protective immunity against systemic and peripheral virusinfection following intra-lymph node immunization with DNA. LCMV titerin spleen (A) and Vacc-G2 vaccinia titers in ovary (B) followingindicated immunization and subsequent viral challenge.

FIG. 9. Growth of transplanted gp33 expressing tumor in mice immunizedby i.ln. injection of gp33 epitope-expressing, or control, plasmid.

FIG. 10. Amount of plasmid DNA detected by real-time PCR in injected ordraining lymph nodes at various times after i.ln. of i.m. injection,respectively.

FIG. 11. Average % supercoiled DNA in formulations 1-9 over 7 days.

DETAILED DESCRIPTION OF THE INVENTION

Method of Treatment

One aspect of this invention is a method for inducing or sustaining aspecific immunological response (i.e., a CTL response) in an animal thathas a disease (or predisposition to a disease) in which the animal'simmune system may attack the disease with a natural CTL response. Theresponse and diseases are discussed in greater detail hereinafter. Themethod has particular value for treating an animal having a malignanttumor in order to inhibit the growth of the tumor or for treating achronic infectious disease such as hepatitis or AIDS.

The method, along with other aspects of the invention, is useful in ananimal having an immune system that includes a lymphatic system. Thisgenerally includes vertebrates, specifically mammals and particularlyhumans. Thus, this invention will find use in treating humans of allages as well as in treating animals, i.e. in veterinary uses. Theinvention may be used for treating livestock such as cattle, sheep,pigs, goats, and the like or for treating household pets such as dogs,cats, rabbits, hamsters, mice, rats, and the like. The primary use willbe for treating humans that are in need of having a specificimmunological response sustained for treatment of a disease such ascancer or chronic infections.

A key aspect of this invention is the delivery of an appropriate antigento the lymphatic system of the animal being treated and sustaining thedelivery over time. This is based in part on the observation that astrong induction and a sustained CTL response require ongoing antigenicstimulation of the lymphatic system. In a human, the lymphatic systemincludes lymph, lymphocytes, lymph vessels, lymph nodes, tonsils, thespleen, the thymus gland, and bone marrow. The lymphatic system performsthree basic functions. First, it helps maintain fluid balance in thetissues. Approximately 30 L of fluid pass from the blood capillariesinto the interstitial spaces each day, whereas only 27 L pass from theinterstitial spaces back into the blood capillaries. If the extra 3 L ofinterstitial fluid were to remain in the interstitial spaces, edemawould result, causing tissue damage and eventual death. These 3 L offluid (i.e. lymph) enter the lymph capillaries, then passes through thelymph vessels to return to the blood. Lymph is similar in composition toplasma. In addition to water, lymph contains solutes derived from twosources: (1) substances in plasma such as ions, nutrients, gases, andsome proteins pass from blood capillaries into the interstitial spacesto become part of the lymph; and (2) substances derived from cellswithin the tissues such as hormones, enzymes, and waste products arealso found in lymph.

The lymphatic system's second basic function is to absorb fats and othersubstances from the digestive tract. Special lymph vessels calledlacteals are in the lining of the small intestine. Fats enter into thelacteals and pass through the lymph vessels to the venous circulation.The lymph passing through these capillaries has a milky appearancebecause of its fat content, and it is called chyle.

The third basic function of the lymphatic system is to act as part ofthe body's defense system. The lymph nodes filter lymph, and the spleenfilters blood, removing microorganisms and other foreign substances.This third function is the function most important to this invention inthat the antigen must be delivered to the lymph system at a levelsufficient to elicit the desired, specific immunological response in theanimal. FIG. 5 is a schematic representation of a human lymphatic systemshowing the major lymphatic organs and vessels.

As hereinbefore mentioned, the present invention relates to a method ofinducing or sustaining a specific immunological response (particularly aCTL response) to an antigen in an animal over time. The method comprisesdelivering the antigen to the animal in a manner that delivers theantigen into the lymphatic system of an animal to sustain the desiredresponse over time. Generally this is done by establishing a mechanismto transfer an antigen from a reservoir to the animal's lymphatic systemon a regular basis over time. The antigen may be delivered by a varietyof methods that target intralymphatic presentation, includingsubcutaneous injection, direct injection into the lymphatic system by anantigen delivery vehicle that is implanted, preferably at or near alymphatic organ, or by an antigen delivery vehicle that is external tothe animal but contains a means (e.g. a needle or catheter) to deliverthe antigen into the lymphatic system. By this method one can avoidmultiple ongoing injections and can also avoid the use of includingprofessional antigen-presenting cells in the composition held in thereservoir.

The method of this invention can be viewed as inducing CTL immuneresponse by providing high continuous local concentrations of antigen,which otherwise is quickly removed and degraded from the body afterbolus injection. Potent activation of CD8+T cells requires signalingthrough the T cell receptor (TCR) in a manner that is dependent on bothquantitative and qualitative factors. Quantitative factors refer to thenumber of TCRs engaged by peptide-MHC complexes. Qualitativeconsiderations include the duration of engagement of the TCR bypeptide-MHC complexes, with specific peptide-MFIC complexes. Sustainedregular deliveries of antigen allows optimal conditions to beestablished for inducing CD8+ T cells.

The antigen is delivered to the animal so that the antigen is present inthe animal's lymphatic system on a sustained basis over a period oftime. That is to say, it is delivered in such a way that the presence ofthe antigen is maintained over the period of time in the animal'slymphatic system. Thus, the antigen is delivered to the animal on aregular basis, i.e. the antigen is delivered regularly withoutsignificant interruption over the period of time. This regular deliveryis achieved by the constant delivery of the antigen at low levelsdirectly to the lymphatic system using an external device or animplantable device, as discussed hereinafter. Alternatively, the antigencan be delivered at higher levels to the animal by subcutaneousinjection with indirect absorption or equilibration with the lymphsystem. Delivery on a regular basis is meant to include intermittent(stopping and transmitting at intervals) as well as continuous(transmitting without interruption) delivery. In intermittent delivery,the times transmission is stopped will not be enough to reduce the levelof antigen in the animal's lymphatic system to eliminate the desiredspecific immunological response. Thus, the antigen may be delivered inpulses or small doses over time.

Preferably, the sustained delivery is achieved by the positioning of ameans of delivery so that the animal being treated does not have toreceive multiple injections of the antigen, but instead has only oneinsertion of the means for delivery, e.g. an insertion of a catheter orneedle for infusion of a suitable antigen-containing composition or thesurgical implantation of an implantable device that releases anappropriate, antigen-containing composition on a sustained basis.

The period of time over which the antigen will be released will be atime sufficient to induce and maintain the desired specificimmunological response, e.g. to maintain a CTL response, and in the caseof an animal with a tumor or infection, at a level sufficient tostimulate the immune system to attack the tumor and inhibit its growthor to attack the infection. Generally, this period of time may vary froma few days, e.g. a week, to a year or more. Preferably, the treatment,i.e. sustained delivery of the antigen, will extend for at least sevendays and no more than six months. It has been found that the CTLresponse is induced by administration for at least seven days. Todetermine the period of time, the attending physician will evaluate,i.e., the severity of the condition, the strength of the patient, theantigenic response (e.g., the level of CD8+ cells measurable in thepatient's system), the presence of toxic effects, and other factorsknown to one of skill in the art. Ultimately the time for sustaineddelivery in a cancer patient will be that necessary for improvement inthe patient as evidenced by reduction in the size of the tumor, the rateof growth of the tumor, and/or the improvement in the overall health ofthe patient being treated. In the treatment of infectious diseases thetreatment is continued until the health of the patient improvessufficiently to stop treatment.

The underlying immunological rationale for the utility of this inventionarises from certain immunological considerations, The immune system hasevolved to protect the host from microbial infection. CD4+ T cellstogether with B cells are the main components of the immune systemhumoral effector arm, which is crucial to eliminate extracellularpathogens or toxins. In contrast, the CD8+ T cell arm of the immunesystem is mainly responsible for eliminating intracellular pathogens,i.e. most importantly viruses, either via cytokine release or bycytotoxic activity. It is now emerging that these most efficient “killercells” of the immune system would best serve as the primary effectorcells in tumor immunotherapy. An object of this invention is to mount adisease-specific CTL response (CD8+ T cell response) against the diseaseand sustain it over time, e.g., a tumor specific or microbial specificCTL response.

CD8+ T cells recognize antigenic oligopeptides presented on HLA class Imolecules of target cells, e.g., tumor cells. The sequences of manyHLA-A1 and HLA-A2 presented tumor and pathogen specific antigen peptideshave recently been characterized. These peptides may be used in thisinvention to induce, e.g., a melanoma-specific CD8+ T cell response.These peptides are discussed hereinafter.

In contrast to viral infection, class I-binding oligopeptides show onlylow immunogenicity. Most viruses induce peak CD8+ T cell responsesaround 7-10 days after systemic spread. This invention aims at enhancingthe immunogenecity of class I binding oligopeptides by sustained,regular release of peptide into a lymphatic system and continued releaseinto the lymphatic system.

In contrast to antibody-mediated B cell memory, which is long lived, Tcell memory appears to be short lived or non-existent. In accordancewith this invention, maintenance of functional T cell memory depends onpersistence of antigen through continued, regular administration of thedesired antigen. Having made this invention and looking at past conceptsthat might support this underlying rationale, some evidence includes theobservation that delayed type hypersensitivity (DTH) of the tuberculintype (the only functional test for T cell memory in humans), can beelicited only in granulomatous disease, such as tuberculosis (tuberculintest), leprosy (lepromin test), brucellosis (brucellin test),sarcoidosis (Kveim test), Histoplasmosis (histoplasimin test) etc., butno such test could be established for non-granulomatous infectiousdisease. A factor that all granulomatous diseases have in common, isthat the antigen persists within the granuloma—professional antigenpresenting cells can use this reservoir to continuously restimulatespecific T cells in lymphoid organs. In mice models (see Example 3) itis demonstrated that maintenance of functional CD8+T cell memory wasstrictly dependent on continuous antigenic restimulation.

To determine whether a CTL response is obtained in an animal beingtreated in accordance with this invention, one measures the level ofCD8+ cells (i.e. CTL) present in the blood or lymphatic organs such asthe spleen or lymph nodes. This determination is done by first measuringthe level of CD8+ cells before performing the method of this inventionand measuring the level during treatment, e.g. at 7, 10, 20, 40 days,etc. The level or strength of the CD8+ (CTL) response can be assessed invivo or in vitro. In humans, there exists so far only one in vivo testto measure CD8+ T cell responses, which is a skin test. In this skintest, HLA class I binding peptides are injected, intradermally (such asdescribed in Jäger, E. et al. Granulocyte-macrophage-colony-stimulatingFactor Enhances Immune Responses To Melanoma-associated Peptides in vivoInt. J Cancer 67, 54-62 (1996)). If a CTL response is present, thesecells will recognize and attack peptide pulsed dermal cells, causing alocal inflammatory reaction either via cytokine release or the cytotoxicmechanism (Kündig, T. M., Althage, A., Hengartner, H. & Zinkemagel, R.M. A skin test to assess CD8+ cytotoxic T cell activity. Proc. Natl.Acad Sci. USA 89:7757-776 (1992)). This inflammatory reaction can bequantified by measuring the diameter of the local skin rash and/or bymeasuring the diameter of the infiltrate (i.e., the swelling reaction).As an alternative to the injection of soluble free peptide, theHLA-class I binding peptide can also be injected intradermally in abound form, e.g., bound to extracorporally derived dendritic cells. Inother mammals, additional, although experimental, in vivo tests toassess CD8+ T cell responses exist. For example, in a mouse model, CD8+T cell responses can be measured by challenge infection with a vacciniarecombinant virus expressing the peptide used for immunization. Whilenaïve mice succumb to the infection with the vaccina recombinant virus,mice with preexisting CD8+ T cell immunity against the peptide epitopeexpressed by the vaccinia recombinant virus, are immune to reinfection.The level of immunity to reinfection can be quantified as the factor ofreduction of the vaccinia virus titer recovered from mouse organs afterchallenge infection (Bachmann, M. F. & Kundig, T. M. In vitro vs. invivo assays for the assessment of T- and B-cell function. Curr. Opin.Immunol. 6, 320-326 (1994)). For example, 5 days after challengeinfection, a typical vaccinia recombinant virus titer recovered from amouse ovary would be around 10⁷ pfu per ovary, whereas the vacciniarecombinant virus titer in a mouse with a preexisting CD8+ T cellresponse against the recombinant gene product would for example bearound 10³ pfu per ovary. Such a 10,000 fold-reduction in virus titerreflects biologically significant preexisting CD8+ T cell activityagainst the recombinant gene product.

The level of CD8+ T cell responses can also be quantified in vitro, byestimating the number of CD8+ T cells specific for the antigenic peptidein question. In a naïve mammal the so called “frequency”, i.e., thenumber of specific CD8+ T cells divided by the number of non-specificwhite blood cells, is less than 10⁻⁶. After successful immunization, thefrequency increases due to proliferation of specific T cells. During anacute viral infection, for example, the frequency of specific CD8+ Tcells may rise to 10⁻². Then, after elimination of the virus, thefrequency of specific CD8+ T cells usually drops to a “memory” level ofaround 10⁻⁴. Thus, the CD8+ T cell response can be quantified bymeasuring the frequency of specific CD8+ T cells. The higher thefrequency, the stronger the response. The classical assays used tomeasure the frequency of specific CD8+ T cells are based on limitingdilution cell culture techniques, as described in detail by Kündig, T.M. et al. (On the role of antigen in maintaining cytotoxic T cellmemory. Proceedings of the National Academy of Sciences of the UnitedStates of America 93, 9716-972′) (1996)). A novel approach to estimatethe frequency of specific CD8+ T cells is to construct soluble class IMHC (for use in mice) or HLA molecules (for use in humans) with apeptide bound to their groove, so that the specific T cell receptorswill bind to these complexes. These complexes can be labeled fordetection, for example, with a fluorescent substance, allowing fordetection by flow cytometry.

One current procedure to render peptides immunogenic is to inject themin context with “nature's most potent adjuvant”, i.e., professionalantigen presenting cells (APCs) such as dendritic cells (DCs),(Steinmann, R. M., The dendritic cells system and its role inimmunogenicity, Annual Review of Immunology 9, 271-96 (1991)). DCs arethe most potent APCs of the immune system. They can now be cultured invitro by adding granulocyte macrophage colony stimulating factor(GM-CSF) and tumor necrosis factor alpha (TNF-alpha) or interleukin-4(IL-4) to progenitors isolated from the blood of patients or mice(Inaba, K. et al., Identification of proliferating dendritic cellprecursors in mouse blood, Journal of Experimental Medicine 175,1157-1167 (1992)). Large numbers of DCs can then be pulsed with tumorspecific antigen peptides and are injected back into the patient, wherethey migrate into lymphatic organs to induce T cell responses (Young, J.W. & Inaba, K., Dendritic Cells As Adjuvants For Class I MajorHistocompatibility Complex-restricted Anti-tumor Immunity, Journal ofExperimental Medicine 183, 7-11 (1996)). An object of this invention isto circumvent the time-consuming, labor intensive procedure of culturingDCs after isolation of DC progenitors and deliver the antigen to thelymphatic system free of APCs such as DCs. The method of this invention,i.e., the sustained, regular delivery of antigen into a lymphatic organ,allows sufficiently high local concentrations of antigen inside thelymphatic organ, such that professional antigen presenting cells, e.g.,dendritic cells, can be loaded with peptide in vivo. This can be viewedas a method of loading antigen presenting cells (dendritic cells) invivo for inducing a CTL response.

The method of the present invention is clearly advantageous over theprior art methods for inducing a CTL response against a tumor or virus.For example, the present invention does not require repetitiveimmunizations to effect for prolonged anti-tumor immunotherapy. Thesustained delivery of the antigen maintains the CTL response that couldultimately afford a prolonged aggressive posture of CTL against tumorcells, more thorough eradication, and protection against recurrenceduring the vaccine treatment. In the absence of antigen, CTL that haveundergone primary activation soon cease to recirculate through the body,soon finding their way to the spleen where they become quiescent. SinceCTL must immediately deliver a lethal hit, their residence in the spleenprecludes an active role in protection against infections or tumorgrowth at distant sites in the body. The controlled release of antigenrecognized by CTL in this invention circumvents this outcome as antigendelivery is maintained. Sustained released antigen delivery to thelymphatic system by this invention solves two major problems: itprovides for potent CTL stimulation that takes place in the milieu ofthe lymphoid organ, and it sustains stimulation that is necessary tokeep CTL active, cytotoxic and recirculating through the body.

Another fundamental improvement of the present method over prior art isthat it facilitates the use of inherently non-immunogenic peptideantigens for CTL stimulation without the combined use of conventionaladjuvants. This is very beneficial as most experimental adjuvants aretoxic and poorly suited for use in humans. In addition adjuvantsstimulate the TH2-type humoral immune response that negatively affectsthe CTL response. Further, since conventional adjuvants are notrequired, only the minimal antigenic epitope for a CTL response isrequired in the formulation.

An additional advantage to the method of the present invention, where itembodies the use of mechanical delivery systems, is that the antigendelivery can be stopped if any adverse immunological effects areobserved, For example, in vaccines against melanoma, CTL have beeninduced to attack not only malignant melanocytes but also healthytissue, causing “vitiligo.” The ability to discontinue a CTL vaccine atany time is a significant advance in vaccine safety. Peptides have ashort half-life due to catabolism in the liver. Therefore, thestimulation-effect falls soon after cessation of delivery.

As pointed out before, the method of this invention has two parts: (1)inducing an increased CTL response and (2) maintaining the response. Theinducing and maintaining may be performed using the same device, asdiscussed hereinafter, or the inducing may be done separately, e.g., bya separate injection of an antigen then following up with sustaineddelivery of the antigen over time to maintain the response.

Diseases Treated According to the Invention

In general, this invention is useful for treating an animal having (orbeing predisposed to) any disease to which the animal's immune systemmounts a cell-mediated response to a disease-related antigen in order toattack the disease. Thus, the type of disease may be a malignant tumoror a chronic infectious disease caused by a bacterium, virus, protozoan,helminth, or other microbial pathogen that enters intracellularly and isattacked, i.e., by the cytotoxic T lymphocytes, In addition, theinvention is useful for treating an animal that may be at risk ofdeveloping such diseases.

Malignant Tumors

In a mature animal, a balance usually is maintained between cell renewaland cell death in most organs and tissues. The various types of maturecells in the body have a given life span; as these cells die, new cellsare generated by the proliferation and differentiation of various typesof stem cells. Under normal circumstances, the production of new cellsis so regulated that the numbers of any particular type of cell remainconstant. Occasionally, though, cells arise that are no longerresponsive to normal growth-control mechanisms. These cells give rise toclones of cells that can expand to a considerable size, producing atumor, or neoplasm. A tumor that is not capable of indefinite growth anddoes not invade the healthy surrounding tissue extensively is benign. Atumor that continues to grow and becomes progressively invasive ismalignant; the term cancer refers specifically to a malignant tumor. Inaddition to uncontrolled growth, malignant tumors exhibit metastasis; inthis process, small clusters of cancerous cells dislodge from a tumor,invade the blood or lymphatic vessels, and are carried to other tissues,where they continue to proliferate. In this way a primary tumor at onesite can give rise to a secondary tumor at another site. The methods,devices and articles of manufacture discussed herein are useful fortreating animals having malignant tumors.

Malignant tumors treated according to this invention are classifiedaccording to the embryonic origin of the tissue from which the tumor isderived. Carcinomas are tumors arising from endodermal or ectodermaltissues such as skin or the epithelial lining of internal organs andglands. A melanoma is a type of carcinoma of the skin for which thisinvention is particularly useful. Sarcomas, which arise less frequently,are derived from mesodermal connective tissues such as bone, fat, andcartilage. The leukemias and lymphomas are malignant tumors ofhematopoietic cells of the bone marrow. Leukemias proliferate as singlecells, whereas lymphomas tend to grow as tumor masses. The malignanttumors may show up at numerous organs or tissues of the body toestablish a cancer. The types of cancer that can be treated inaccordance with this invention include the following: bladder, brain,breast, cervical, colo-rectal, esophageal, kidney, liver, lung,nasopharangeal, pancreatic, prostate, skin, stomach, uterine, and thelike. The present invention is not limited to the treatment of anexisting tumor or infectious disease but can also be used to prevent orlower the risk of developing such diseases in an individual, ie., forprophylactic use. Potential candidates for prophylactic vaccinationinclude individuals with a high risk of developing cancer, i.e., with apersonal or tuminal history of certain types of cancer.

The incidence of skin cancer has increased substantially over the lastdecades. Lifetime analysis indicates that around 1/1500 humans born in1935, 1/600 born in 1960, 1/100 born in 1990 and a projected 1/75 humansborn in the year 2000 will have melanoma in their lifetime. Surgicalexcision usually cures melanoma. However, even small looking lesions mayhave already metastasized at the time of diagnosis. The prognosis ofmetastasized melanoma is very poor and correlates with the thickness ofthe primary tumor and with its localization.

The current treatment of malignant melanoma aims at surgical removal ofthe primary tumor. If metastases are present, chemotherapy andbiological response modifiers are additionally used. However, patientswith stage IV malignant melanoma are almost invariably incurable andtreatments are palliative. Patients with Stage IV malignant melanomahave a median survival time of approximately one year and only a 10%chance of long-term survival. There is at present no generally acceptedstandard therapy for metastatic melanoma. Objective response rates tomono- or polychemotherapy are low in comparison with other tumors,reaching no more than 15-35%. An improved treatment outcome in stage IVmalignant melanoma seems unachievable either by chemotherapeuticcombinations or by increasing doses to levels where autologous bonemarrow transplantation becomes necessary. The method of this inventionis useful for treating malignant melanoma, even at Stage IV.

Infectious Diseases

Infectious diseases, which have plagued animal populations (particularlyhumans) throughout-history, still cause millions of deaths each year.The infectious diseases that can be treated using this invention includethose caused by pathogens such as bacteria, viruses, protozoa,helminths, and the like. These diseases include such chronic diseasessuch as acute respiratory infections, diarrheal diseases, tuberculosis,malaria, hepatitis (hepatitis A, B C, D, E, F virus), measles,mononucleosis (Epstein-Barr virus), whooping cough (pertussis), AIDS(human immunodeficiency virus I & 2), rabies, yellow fever, and thelike. Other diseases caused by human papilloma virus or various strainsof virus are treatable by this method.

In some instances, the mammal, in particular human, can be treatedprophylactically, such as when there may be a risk of developingdisease. An individual travelling to or living in an area of endemicinfectious disease may be considered to be at risk and a candidate forprophylactic vaccination against the particular infectious agent. Forexample, the CTL response can be induced in a human expecting to enter amalarial area and/or while in the malarial area by using a CTL-inducing,-malaria-specific antigen to lower the risk of developing malaria.Preventative treatment can be applied to any number of diseasesincluding those listed above, where there is a known relationshipbetween the particular disease and a particular risk factor, such asgeographical location or work environment.

Antigens Useful in the Invention

An antigen useful in this invention is one that stimulates the immunesystem of a mammal having a malignant tumor or infectious disease toattack the tumor and inhibit its growth or to destroy the pathogencausing the disease. Thus, the antigen used in the invention is matchedto the specific disease found in the animal being treated. In thisregard the antigen may be said to induce a CTL response (also referredto as a cell-mediated immune response), i.e. a cytotoxic reaction by theimmune system that results in lysis of the target cells (e.g., themalignant tumor cells or pathogen-infected cells).

To determine whether an antigen is matched to a particular patient,whether human or other animal, the tissue type of the patient is firstdetermined. If human, the tissue must demonstrate the appropriate humanleukocyte antigen (HLA) capable of binding and displaying the antigen toCTL. It is preferable that the HLA typing be performed, on the targetcells, since a significant portion of tumors escape immune detection bydownregulating expression of HLA. Therefore HLA expression on normalcells of the patient does not necessarily reflect that found on tumorcells in their body. A tumor from a patient is also screened todetermine if he or she expresses the antigen that is being used in thevaccine formulation. Immunohistochemistry and/or polymerase chainreaction (PCR) techniques both can be used to detect antigen in thetumor cells. Immunchistochemistry offers the advantage in that it stainsa cross-section of tumor in a slide preparation, allowing investigatorsto observe the antigen expression pattern in cross-section of tumor,which is typically heterogeneous for antigen expression. PCR has theadvantage of not requiring specific monoclonal antibodies for stainingand is a fast and powerful technique. In addition, PCR can be applied insitu. Ideally, both immunohistochemical and PCR methods should becombined when assessing antigen expression in tumors. While the antigencompositions useful in this invention are designed to include the mostcommonly expressed tumor antigens (as discussed hereafter), not alltumors will express the desired antigen(s). Where a tumor fails toexpress the desired antigen, the patient is excluded for considerationfor that particular antigen composition. Thus, an aspect of thisinvention is a process for preparing a device useful for providing asustained CTL response over time by matching a subject's antigenspecific to the tumor or pathogen in the subject, preparing aphysiologically-acceptable composition of the antigen so matched, andcombining the composition in a suitable delivery device as discussed inhereinafter.

Immune activation of CD8+ T cells generates a population of effectorcells with lytic capability called cytotoxic T lymphocytes, or CTL.These effector cells have important roles in the recognition andelimination of malignant cells and pathogens. In general, CTL are CD8+and are therefore class I MHC restricted, although in rare instancesCD4+ class II—restricted T cells have been shown to function as CTL.Since virtually all nucleated cells in the body express class I MHCmolecules, CTL can recognize and eliminate almost any altered body cell.CD8+T cells recognize antigen presented on HLA class I molecules oftumor cells through T cell receptors.

The CTL-mediated immune response can be divided into two phases,reflecting different aspects of the cytotoxic T-cell response. The firstphase involves the activation and differentiation of T_(c) (CD8+) cellsinto functional effector CTLs. In the second phase, CTLs, recognizeantigen—class I MHC complexes on specific target cells, initiating asequence of events that culminates in target-cell destruction. Furtherdetailed discussion of the process is found at Chapter 15 of the SecondEdition of “Immunology” by Janis Kuby, W.H. Freeman and Company (1991).

The type of tumor antigen used in this invention may be a tumor-specificantigen (TSA) or a tumor-associated antigen (TAA), A TSA is unique totumor cells and does not occur on other cells in the body. A TAAassociated antigen is not unique to a tumor cell and instead is alsoexpressed on a normal cell under conditions that fail to induce a stateof immunologic tolerance to the antigen. The expression of the antigenon the tumor may occur under conditions that enable the immune system torespond to the antigen. TAAs may be antigens that are expressed onnormal cells during fetal development when the immune system is immatureand unable to respond or they may be antigens that are normally presentat extremely low levels on normal cells but which are expressed at muchhigher levels on tumor cells. TSAs and TAAs can be jointly referred toas TRA or a tumor related antigen.

Tumor antigens useful in the present invention, whether tumor-specificor tumor-associated, must be capable of inducing a CTL-mediated immuneresponse. The presence of tumor antigens that elicit a cell-mediatedresponse has been demonstrated by the rejection of tumors transplantedinto syngeneic recipients; because of this phenomenon, these tumorantigens are referred to as tumor-specific transplantation antigens(TSTAs) or tumor-associated transplantation antigens (TATAs). It hasbeen difficult to characterize tumor transplantation antigens becausethey do not generally elicit an antibody response and therefore theycannot be isolated by immunoprecipitation. Many are peptides that arepresented together with MHC molecules on the surface of tumor cells andhave been characterized by their ability to induce an antigen-specificCTL.

The type of pathogen specific antigen useful in this invention may beshort oligopeptides derived from pathogen proteins. These oligopeptidesmust bind to class I MHC (for use in mice), class I HLA (for use inhumans), or class I molecules of any other mammals. Also, such class Imolecule bound peptides should be recognizable by specific T cellreceptors. Such oligopeptides usually have a length of 8-15 amino acids.Several examples of such pathogen derived oligopeptides, so called Tcell epitopes, are given in Tables I and II.

The tumor antigens and pathogen-specific antigens useful in thisinvention are generally thought to be presented at the surface of anantigen presenting cell (APC) to stimulate the immune system throughclass I molecules of the major histocompatability complex (MHC)interactively with the CD8+ cells.

Antigens useful in the invention are generally protein-based entities ofa molecular weight of up to 100,000 daltons. Appropriate antigensinclude, but are not limited to differentiation antigens, tumor-specificmultilineage antigens, embryonic antigens, antigens of oncogenes andmutated tumor-suppressor genes, unique tumor antigens resulting fromchromosomal translocations, viral antigens, and others that may beapparent presently or in the future to one of skill in the art. It ispreferable that the antigen be a peptide of 8 to 15 amino acids inlength that is an epitope of a larger antigen, i.e. it is a peptidehaving an amino acid sequence corresponding to the site on the largermolecule that is recognized and bound by a particular T-cell receptor.These smaller peptides are available to one of skill in the art byfollowing the teachings of U.S. Pat. No. 5,747,269 to Rarnmensee et al.issued May 5, 1998; U.S. Pat. No. 5,698,396 to Pfreundschuh issued Dec.16, 1997; and PCT Application Numbers PCT/EP95/02593 filed 4 Jul. 1995,PCT/DE96/00351 filed 26 Feb. 1996, all of which are incorporated hereinby reference. Additional approaches to epitope discovery are describedin U.S. Pat. No. 6,037,135 METHODS FOR MAKING HLA BINDING PEPTIDES ANDTHEIR USES and U.S. patent application Ser. No. 09/561,074 entitledMETHOD OF EPITOPE DISCOVERY both of which are incorporated herein byreference in their entirety.

While in the general case the antigen ultimately recognized by a T cellis a peptide, it must be kept in mind that the form of antigen actuallyadministered as the immunogenic preparation need not be a peptide perse. When administered, the epitopic peptide(s) may reside within alonger polypeptide, whether the complete protein antigen, some segmentof it, or some engineered sequence. Included in such engineeredsequences would be polyepitopes and epitopes incorporated into somecarrier sequence such as an antibody or viral capsid protein. Suchlonger polypeptides may include epitope clusters as described in U.S.patent application Ser. No. 09/561,571 entitled “EPITOPE CLUSTERS,”which is incorporated herein by reference in its entirety. The epitopicpeptide, or the longer polypeptide in which it is contained, may be acomponent of a microorganism (e.g. a virus, bacterium, protozoan, etc.),or a mammalian cell (e.g. a tumor cell or antigen presenting cell), orlysates, whole or partially purified, of any of the foregoing. They maybe used as complexes with other proteins, for example heat shockproteins. The epitopic peptide may also be covalently modified, such asby lipidation, or made a component of a synthetic compound, such asdendrimers, multiple antigen peptides systems (MAPS), and polyoximes, ormay be incorporated into liposomes or microshperes, etc. As used in thisdisclosure the term “polypeptide antigen” encompasses all suchpossibilities and combinations. The invention comprehends that theantigen may be a native component of the microorganism or mammaliancell. The antigen may also be expressed by the microorganism ormammalian cell through recombinant DNA technology or, especially in thecase of antigen presenting cells, by pulsing the cell with polypeptideantigen prior to administration. Additionally, the antigen may beadministered encoded by a nucleic acid that is subsequently expressed byAPCs. Finally, whereas the classical class I MHC molecules presentpeptide antigens, there are additional class I molecules which areadapted to present non-peptide macromolecules, particularly componentsof microbial cell walls, including without limitation lipids andglycolipids. As used in this disclosure the term antigen comprehendssuch macromolecules as well. Moreover, a nucleic acid based vaccine mayencode an enzyme or enzymes necessary to the synthesis of such amacromolecule and thereby confer antigen expression on an APC.

A powerful method has been recently developed for identifying newpeptides that are useful in the invention. Genes determined to expressprotein with high exclusivity in tumor cells or microbial cells (e.g.viruses) can be identified using a so called SEREX process, whichinvolves expression cloning using tumor cell libraries and screeningthese libraries against immunoglobulin in patient sera. Over one hundredgenes have recently been identified from tumor biopsies using thisprocess. These genes can now be used in a peptide prediction algorithmdeveloped by Hans-Georg Rammensee. Algorithms have been developed forall major HLA types found in the human population. First the proteinsequence is “translated” based on the gene sequence. The algorithms canpredict peptide epitopes for various HLA types based on the proteinsequence. Since the predicted peptides are indeed predictions and arenot always naturally found on cells, tumor samples are used to confirmthe predicted peptides by actually isolating minute trace peptide fromtumors. Being able to calculate the exact mass of the predicted peptidesallows trace peptide identification using ultrasensitive massspectrophotometry, which can detect peptides in quantities less thatthat which would permit peptide sequencing and identification. Oncethese tumor-associated peptides have been identified they are suitablefor use in the invention, since peptides of a known sequence may besynthesized in large quantities (several grams) providing for sufficientamounts of peptides for use in this invention.

In addition to the imperfection of existing prediction algorithms forMHC binding, some peptides that would be fully capable of binding to MHCmay never be liberated by protoelytic processing from the completeprotein antigen. Methods for evaluating which peptides will be liberatedby proteasomal processing have been developed, e.g. U.S. patentapplication Ser. No. 09/561,074 supra, increasing the efficiency withwhich useful epitopes can be discovered. Moreover, proteasomalprocessing can differ between target cell and APC such that care must betaken in the identification and selection of epitopes and in vaccinedesign so that the vaccine will induce a response that will in factrecognize the target cell. These issues are more fully discussed in U.S.patent application Ser. No. 09/560,465 entitled “EPITOPESYNCHRONIZATION,” which is incorporated herein by reference in itsentirety.

Thus it can be seen that another aspect of this invention is a processfor preparing a composition useful in a device of this invention asdiscussed hereinafter. The process comprises identifying a genedetermined to express a protein with high exclusivity in a tumor ormicrobial cell, cloning cell libraries, screening the libraries againstimmunoglobulin in patient sera, using the algorithm defined in theliterature developed by Hans-George Rammensee to predict an epitope forthe HLA type protein based on the gene sequence, matching the predictedantigen sequence to a patient tumor sample, isolating the matchedantigen, and preparing a composition of the antigen for use in adelivery device as discussed hereinafter.

Examples of large, protein-based antigens include the following:

Differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigenssuch as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressedembryonic antigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumorantigens resulting from chromosomal translocations; such as BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as theEpstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,β-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA,CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM,HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16,TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6,TAG72, TLP, and TPS. These protein-based antigens are known andavailable to those of skill in the art in the literature orcommercially.

Examples of peptide antigens of 8-15 amino acids include those set forthin Table I, Table II, and Table III. Table I sets forth antigens thatare virally derived. The Table shows the virus type, the proteinexpressed by the virus, the amino acid (AA) position on the viralprotein, the AA sequence of the T-cell epitope/MHC ligand, the type ofMHC molecule presenting the antigen, and a reference source. A morecomplete list is provided in the book by Han-Georg Rammensee, JuttaBachmann, and Stefan Stevanovic entitled “MHC Ligands and PeptideMotifs,” Springer-Verlag, Germany, 1997 Landes Bioscience, Austin,Tex.). The reference number given in Table I is the same number (andreference source) given in Table 5.3 of the above Rammensee book, all ofwhich is incorporated herein by reference.

TABLE I Viral epitopes on MHC class 1 molecules T cell epitope AA MHCligand MHC Virus Protein Position (Antigen) molecule Ref. Adenovirus 3E3 9Kd 30-38 LIVIGILIL HLA-A*0201 104 (SEQ. ID NO.:1) Adenovirus 5 EIA234-243 SGPSNTPPEI H2-Db 105 (SEQ. ID NO.:2) Adenovirus 5 ElB 192-200VNIRNCCYI H2-Db 106 (SEQ. ID NO.:3) Adenovirus 5 EIA 234-243 SGPSNIPPEI(T > 1) H2-Db 106 (SEQ. ID NO.:4) CSFV NS 2276-2284 ENALLVALF SLA, 107polyprote (SEQ. ID NO.:5 haplotype in d/d Dengue virus NS3 500-508TPEGIIPTL HLA-B*3501 108, 109 4 (SEQ. ID NO.:6 EBV LMP-2 426-434CLGGLLTMV HLA-A*0201 110 (SEQ. ID NO.:7) EBV EBNA-1 480-484 NIAEGLRALHLA-A*0201 111 (SEQ. ID NO.:8) EBV EBNA-1 519-527 NLRRGTALA HLA-A*0201111 (SEQ. ID NO.:9) EBV EBNA-1 525-533 ALAIPQCRL HLA-A*0201 111 (SEQ. IDNO.:10) EBV EBNA-1 575-582 VLKDAIKDL HLA-A*0201 111 (SEQ. ID NO.:11) EBVEBNA-1 562-570 FMVFLQTHI HLA-A*0201 111 (SEQ. ID NO.:12) EBV EBNA-215-23 HLIVDTDSL HLA-A*0201 111 (SEQ. ID NO.:13) EBV EBNA-2 22-30SLGNPSLSV HLA-A*0201 111 (SEQ. ID NO.:14) EBV EBNA-2 126-134 PLASAMRMLHLA-A*0201 111 (SEQ. ID NO.:15) EBV EBNA-2 132-140 RMLWMANYI HLA-A*0201111 (SEQ. ID NO.:16) EBV EBNA-2 133-141 MLWMANYIV HL.A-A*0201 111 (SEQ.ID NO.:17) EBV EBNA-2 151-159 ILPQGPQTA HLA-A*0201 111 (SEQ. ID NO.:18)EBV EBNA-2 171-179 PLRPTAPTI HLA-A*0201 111 (SEQ. ID NO.:19) EBV EBNA-2205-213 PLPPATLTV HLA-A*0201 111 (SEQ. ID NO.:20) EBV EBNA-2 246-254RMHLPVLHV HLA-A*0201 111 (SEQ. ID NO.:21) EBV EBNA-2 287-295 PMPLPPSQLHLA-A*0201 111 (SEQ. ID NO.:22) EBV EBNA-2 294-302 QLPPPAAPA HLA-A*0201111 (SEQ. ID NO.:23) EBV EBNA-2 381-389 SMPELSPVL HLA-A*0201 111 (SEQ.ID NO.:24) EBV EBNA-2 453-461 DLDESWDYI HLA-A*0201 111 (SEQ. ID NO.:25)EBV BZLF1 43-51 PLPCVLWPV HLA-A*0201 111 (SEQ. ID NO.:26) EBV BZLF1167-175 SLEECDSEL HLA-A*0201 111 (SEQ. ID NO.:27) EBV BZLF1 176-184EIKRYKNRV HLA-A*0201 111 (SEQ. ID NO.:28) EBV BZLF1 195-203 QLLQHYREVHLA-A*0201 111 (SEQ. ID NO.:29) EBV BZLF1 196-204 LLQHYREVA HLA-A*0201111 (SEQ. ID NO.:30) EBV BZLF1 217-225 LLKQMCPSL HLA-A*0201 111 (SEQ. IDNO.:31) EBV BZLF1 229-237 SIIPRTPDV HLA-A*0201 111 (SEQ. ID NO.:32) EBVEBNA-6 284-293 LLDFVRFMGV HLA-A*0201 112 (SEQ. ID NO.:33) EBV EBNA-3464-472 SVRDRLARL HLA-A*0203 113 (SEQ. ID NO.:34) EBV EBNA-4 416-424IVTDFSVIK HLA-A*1101 114, 115 (SEQ. ID NO.:35) EBV EBNA-4 399-408AVFDRKSDAK HLA-A*0201 116 (SEQ. ID NO.:36) EBV EBNA-3 246-253 RYSIFFDYHLA-A24 113 (SEQ. ID NO.:37) EBV EBNA-6 881-889 QPRAPIRPI HLA-B7 117(SEQ. ID NO.:38) EBV EBNA-3 379-387 RPPIFIRRI. HLA-B7 117 (SEQ. IDNO.:39) EBV EBNA-1 426-434 EPDVPPGAI HLA-B7 111 (SEQ. ID NO.:40) EBVEBNA-1 228-236 IPQCRLTPL HLA-B7 111 (SEQ. ID NO.:41) EBV EBNA-1 546-554GPGPQPGPL HLA-B7 111 (SEQ. ID NO.:42) EBV EBNA-1 550-558 QPGPLRESIHLA-B7 111 (SEQ. ID NO.:43) EBV EBNA-1 72-80 R.PQKRPSCI HLA-B7 111 (SEQ.ID NO.:44) EBV EBNA-2 224-232 PPTPLLTVL HLA-B7 111 (SEQ. ID NO.:45) EBVEBNA-2 241-249 TPSPPRMHL HLA-B7 111 (SEQ. ID NO.:46) EBV EBNA-2 244-252PPRMHLPVL HLA-B7 111 (SEQ. ID NO.:47) EBV EBNA-2 254-262 VPDQSMHPLHLA-B7 111 (SEQ. ID NO.:48) EBV EBNA-2 446-454 PPSIDPADL HLA-B7 111(SEQ. ID NO.:49) EBV BZLFI 44-52 LPCVLWPVL HLA-B7 111 (SEQ. ID NO.:50)EBV BZLF1 222-231 CPSLDVDSII HLA-B7 111 (SEQ. ID NO.:51) EBV BZLFI234-242 TPDVLHEDL HLA-B7 111 (SEQ. ID NO.:52) EBV EBNA-3 339-347FLRGRAYGL HLA-B8 118 (SEQ. ID NO.:53) EBV EBNA-3 26-34 QAKWRLQTL HLA-B8113 (SEQ. ID NO.:54) EBV EBNA-3 325-333 AYPLHEQHG HLA-B8 116 (SEQ. IDNO.:55) EBV EBNA-3 158-166 YIKSFVSDA HLA-B8 116 (SEQ. ID NO.:56) EBVLMP-2 236-244 RRRWRRLTV HLA-B*2704 119 (SEQ. ID NO.:57) EBV EBNA-6258-266 RRIYDLIEL HLA-B*2705 119 (SEQ. ID NO.:58) EBV EBNA-3 458-466YPLHEQHGM HLA-B*3501 120 (SEQ. ID NO.:59) EBV EBNA-3 458-466 YPLHEQHGMHLA-B*3503 113 (SEQ. ID NO.:59) HCV NS3 389-397 HSKKKCDEL HLA-B8 145(SEQ. ID NO.:60) HCV env E 44-51 ASRCWVAM HLA-B*3501 146 (SEQ. IDNO.:61) HCV core 27-35 GQIVGGVYL HLA-B*40012 147 protein (SEQ. IDNO.:62) HCV NSI 77-85 PPLTDFDQGW HLA-B*5301 145 (SEQ. ID NO.:63) HCVcore 18-27 LMGYIPLVGA H2-Dd 138 protein (SEQ. ID NO.:64) HCV core 16-25ADLMGYIPLV H2-Dd 148 protein (SEQ. ID NO.:65) HCV NS5 409-424MSYSWTGALVTPC H2-Dd 149 AEE (SEQ. ID NO.:66) HCV NS1 205-213 KHPDATYSRPapa-A06 150 (SEQ. ID NO.:67) HCV-1 NS3 400-409 KLVALGLNAV HLA-A*0201141 (SEQ. ID NO.:68) HCV-1 NS3 440-448 GDFDSVIDC Patr-B16 151 (SEQ. IDNO.:69) HCV-1 env E 118-126 GNASRCWVA Patr-BI6 151 (SEQ. ID NO.:70)HCV-1 NSI 159-167 TRPPLGNWF Patr-B13 151 (SEQ. ID NO.:71) HCV-1 NS3351-359 VPHPNIEEV Patr-B13 151 (SEQ. ID NO.:72) HCV-1 NS3 438-446YTGDFDSVI Patr-B01 151 (SEQ. ID NO.:73) HCV-1 NS4 328-335 SWAIKWEYPatr-A1 1 151 (SEQ. ID NO.:74) HCV-1 NSI 205-213 KHPDATYSR Patr-A04 150(SEQ. ID NO.:75) HCV-1 NS3 440-448 GDFDSVIDC Patr-A04 150 (SEQ. IDNO.:76) HIV gp41 583-591 RYLKDQQLL HLA_A24 152 (SEQ. ID NO.:77) HIVgagp24 267-275 IVGLNKIVR HLA-A*3302 153, 154 (SEQ. ID NO.:78) HIV gagp24262-270 EIYKRWIIL HLA-B8 155, 156 (SEQ. ID NO.:79) HIV gagp24 261-269GEIYKRWII HLA-B8 155, 156 (SEQ. ID NO.:80) HIV gagp17  93-101 EIKDTKEALHLA-B8 155, 157 (SEQ. ID NO.:81) HIV gp41 586-593 YLKDQQLL HLA-B8 158(SEQ. ID NO.:82) HIV gagp24 267-277 ILGLNKIVRMY HLA-B* 1501 153 (SEQ. IDNO.:83) HIV gp41 584-592 ERYLKDQQL HLA-B14 158 (SEQ. ID NO.:84) HIV nef115-125 YHTQGYFPQWQ HLA-B17 159 (SEQ. ID NO.:85) HIV nef 117-128TQGYFPQWQNYT HLA-B17 159 (SEQ. ID NO.:86) HIV gp120 314-322 GRAFVTlGKHLA-B*2705 160, 184 (SEQ. ID NO.:87) HIV gagp24 263-271 KRWIILGLNHLA-B*2702 161 (SEQ. ID NO.:88) HIV nef 72-82 QVPLRPMTYK HLA-B*3501 159(SEQ. ID NO.:89) HIV nef 117-125 TQGYFPQWQ HLA-B*3701 159 (SEQ. IDNO.:90) HIV gagp24 143-151 HQAISPRTI, HLA-Cw*0301 162 (SEQ. ID NO.:91)HIV gagp24 140-151 QMVHQAISPRTL HLA-Cw*0301 162 (SEQ. ID NO.:92) HIVgp120 431-440 MYAPPIGGQI H2-Kd 163 (SEQ. ID NO.:93) HIV gp160 318-327RGPGRAFVTI H2-Dd 164, 165 (SEQ. ID NO.:94) HIV gp120 17-29 MPGRAFVTIH2-Ld 166, 167 (SEQ. ID NO.:95) HIV-1 RT 476-484 ILKEPVHGV HLA-A*0201168, 169 (SEQ. ID NO.:96) HIV-1 nef 190-198 AFHHVAREL HLA-A*0201 170(SEQ ID NO.:97) HIV-1 gpI60 120-128 KLTPLCVTL HLA-A*0201 171 (SEQ IDNO.:98) HIV-1 gp]60 814-823 SLLNATDIAV HLA-A*0201 171 (SEQ ID NO.:99)HIV-1 RT 179-187 VIYQYMDDL HLA-A*0201 172 (SEQ ID NO.:100) HIV-1 gagp 1777-85 SLYNTVATL HLA-A*0201 173 (SEQ ID NO.:101) HIV-1 gp160 315-329RGPGRAFVT1 HLA-A*0201 174 (SEQ ID NO.:102) HIV-1 gp41 768-778RLRDLLLIVTR HLA-A3 175, 178 (SEQ ID NO.:103) HIV-1 nef 73-82 QVPLRPMTYKHLA-A3 176 (SEQ ID NO.:104) HIV-1 gp120 36-45 TVYYGVPVWK HLA-A3 177(SEQ. ID NO.:105) HIV-1 gagp17 20-29 RLRPGGKKK HLA-A3 177 (SEQ. IDNO.:106) HIV-1 gp120 38-46 VYYGVPVWK HLA-A3 179 (SEQ. ID NO.:107) HIV-1nef 74-82 VPLRPMTYK HLA-a*1101 114 (SEQ. ID NO.:108) HIV-1 gagp24325-333 AIFQSSMTK HLA-A*1101 114 (SEQ. ID NO.:109) HIV-1 nef 73-82QVPLRPMTYK HLA-A*1101 180 (SEQ. ID NO.:104) HIV-1 nef 83-94 AAVDLSHFLKEKHLA-A*1101 159 (SEQ. ID NO.:110) HIV-1 gagp24 349-359 ACQGVGGPGGHKHLA-A*1101 181 (SEQ. ID NO.:111) HIV-1 gagp24 203-212 ETINEEAAEW HLA-A25182 (SEQ. ID NO.:112) HIV-1 nef 128-137 TPGPGVRYPL HLA-B7 159 (SEQ. IDNO.:113) HIV-1 gagp 17 24-31 GGKKKYKL HLA-B8 183 (SEQ. ID NO.:114) HIV-1gp120  2-10 RVKEKYQHL HLA-B8 181 (SEQ. ID NO.:115) HIV-1 gagp24 298-306DRFYKTLRA HLA-B 14 173 (SEQ. ID NO.:116) HIV-1 NEF 132-147 GVRYPLTFGWCYKHLA-B18 159 LVP (SEQ. ID NO.:117) HIV-1 gagp24 265-24  KRWIILGLNKHLA-B*2705 184, 153 (SEQ. ID NO.:118) HIV-1 nef 190-198 AFHHVARELHLA-B*5201 170 (SEQ. ID NO.:97) EBV EBNA-6 335-343 KEHVIQNAF HLA-B44 121(SEQ. ID NO.:119) EBV EBNA-6 130-139 EENLLDFVRF HLA-B*4403 122 (SEQ. IDNO.:120) EBV EBNA-2 42-51 DTPLIPLTIF HLA-B51 121 (SEQ. ID NO.:121) EBVEBNA-6 213-222 QNGALAINTF HLA-1362 112 (SEQ. ID NO.:122) EBV EBNA-3603-611 RLRAEAGVK HLA-A3 123 (SEQ. ID NO.:123) HBV sAg 348-357GLSPTVWLSV HLA-A*0201 124 (SEQ. ID NO.:124) HBV SAg 335-343 WLSLLVPFVHLA-A*0201 124 (SEQ. ID NO.:125) HBV cAg 18-27 FLPSDFFPSV HLA-A*0201125, 126 (SEQ. ID NO.:126) 127 HBV cAg 18-27 FLPSDFFPSV HLA-A*0202 127(SEQ. ID NO.:126) HBV cAg 18-27 FLPSDFFPSV HLA-A*0205 127 (SEQ. IDNO.:126) HBV cAg 18-27 FLPSDFFPSV HLA-A*0206 127 (SEQ. ID NO.:126) HBVpol 575-583 FLLSLGIHL HLA-A*0201 128 (SEQ. ID NO.:127) HBV pol 816-824SLYADSPSV HLA-A*0201 128 (SEQ. ID NO.:128) HBV pol 455-463 GLSRYVARLHLA-A*0201 128 (SEQ. ID NO.:129) HBV env 338-347 LLVPFVQWFV HLA-A*0201129 (SEQ. ID NO.:130) HBV pol 642-650 ALMPLYACI HLA-A*0201 129 (SEQ. IDNO.:131) HBV env 378-387 LLPIFFCLWV HLA-A*0201 129 (SEQ. ID NO.:132) HBVpol 538-546 YMDDVVLGA HLA-A*0201 129 (SEQ. ID NO.:133) HBV env 250-258LLLCLIFLL HLA-A*0201 130 (SEQ. ID NO.:134) HBV env 260-269 LLDYQGMLPVHLA-A*0201 130 (SEQ. ID NO.:135) HBV env 370-379 SIVSPFIPLL HLA-A*0201130 (SEQ. ID NO.:136) HBV env 183-191 FLLTRILTI HLA-A*0201 130 (SEQ. IDNO.:137) HBV cAg 88-96 YVNVNMGLK HLA-A* 1101 131 (SEQ. ID NO.:138) HBVcAg 141-151 STLPETTVVRR HLA-A*3101 132 (SEQ. ID NO.:139) HBV cAg 141-151STLPETTVVRR HLA-A*6801 132 (SEQ. ID NO.:139) HBV cAg 18-27 FLPSDFFPSVHLA-A*6801 127 (SEQ. ID NO.:126) HBV sAg 28-39 IPQSLDSWWTSL H2-Ld 133(SEQ. ID NO.:140) HBV cAg  93-100 MGLKFRQL H2-Kb 134 (SEQ. ID NO.:141)HBV preS 141-149 STBXQSGXQ HLA-A*0201 135 (SEQ. ID NO.:142) HCMV gp B618-628 FIAGNSAYEYV HLA-A*0201 124 (SEQ. ID NO.:143) HCMV E1 978-989SDEEFAIVAYTL HLA-B18 136 (SEQ. ID NO.:144) HCMV pp65 397-411DDVWTSGSDSDEE HLA-b35 137 LV (SEQ. ID NO.:145) HCMV pp65 123-131IPSINVHHY HLA-B*3501 136 (SEQ. ID NO.:146) HCMV pp65 495-504 NLVPMVATVOHLA-A*0201 137 (SEQ. ID NO.:147) HCMV pp65 415-429 RKTPRVTOGGAMA HLA-B7137 GA (SEQ. ID NO.:148) HCV MP 17-25 DLMGYIPLV HLA-A*0201 138 (SEQ. IDNO.:149) HCV MP 63-72 LLALLSCLTV HLA-A*0201 139 (SEQ. ID NO.:150) HCV MP105-112 ILHTPGCV HLA-A*0201 139 (SEQ. ID NO.:151) HCV env E 66-75QLRRHIDLLV HLA-A*0201 139 (SEQ. ID NO.:152) HCV env E 88-96 DLCGSVFLVHLA-A*0201 139 (SEQ. ID NO.:153) HCV env E 172-180 SMVGNWAKV HLA-A*0201139 (SEQ. ID NO.:154) HCV NSI 308-316 HLIIQNIVDV HLA-A*0201 139 (SEQ. IDNO.:155) HCV NSI 340-348 FLLLADARV HLA-A*0201 139 (SEQ. ID NO.:156) HCVNS2 234-246 GLRDLAVAVEPVV HLA-A*0201 139 (SEQ. ID NO.:157) HCV NSI 18-28SLLAPGAKQNV HLA-A*0201 139 (SEQ. ID NO.:158) HCV NSI 19-28 LLAPGAKQNVHLA-A*0201 139 (SEQ. ID NO.:159) HCV NS4 192-201 LLFNILGGWV HLA-A*0201129 (SEQ. ID NO.:160) HCV NS3 579-587 YLVAYQATV HLA-A*0201 129 (SEQ. IDNO.:161) HCV core 34-43 YLLPRRGPRL HLA-A*0201 129 protein (SEQ. IDNO.:162) HCV MP 63-72 LLALLSCLTI HLA-A*0201 129 (SEQ. ID NO.:163) HCVNS4 174-182 SLMAFTAAV HLA-A*0201 140 (SEQ. ID NO.:164) HCV NS3 67-75CINGVCWTV HLA-A*0201 140 (SEQ. ID NO.:165) HCV NS3 163-171 LLCPAGHAVHLA-A*0201 141 (SEQ. ID NO.:166) HCV NS5 239-247 ILDSFDPLV HLA-A*0201141 (SEQ. ID NO.:167) HCV NS4A 236-244 ILAGYGAGV HLA-A*0201 142 (SEQ. IDNO.:168) HCV NS5 714-722 GLQDCTMLV HLA-A*0201 142 (SEQ. ID NO.:169) HCVNS3 281-290 TGAPVTYSTY HLA-A*0201 143 (SEQ. ID NO.:170) HCV NS4A 149-157HMWNFISGI HLA-A*0201 144 (SEQ. ID NO.:171) HCV NS5 575-583 RVCEKMALYHLA-A*0201-A3 145 (SEQ. ID NO.:172) HCV NS1 238-246 TINYTIFK HLA-A*1101145 (SEQ. ID NO.:173) HCV NS2 109-116 YISWCLWW HLA-A23 145 (SEQ. IDNO.:174) HCV core 40-48 GPRLGVRAT HLA-B7 145 protein (SEQ. ID NO.:175)HIV-1 gp120 380-388 SFNCGGEFF HLA-Cw*0401 185 (SEQ. ID NO.:176) HIV-1 RT206-214 TEMEKEGKI H2-Kk 186 (SEQ. ID NO.:177) HIV-1 p17 18-26 KIRLRPGGKHLA-A*0301 187 (SEQ. ID NO.:178) HIV-1 P17 20-29 RLRPGGKKKY HLA-A*0301188 (SEQ. ID NO.:179) HIV-I RT 325-333 AIFQSSMTK HLA-A*0301 188 (SEQ. IDNO.:180) HIV-1 p17 84-92 TLYCVHQRI HLA-A11 188 (SEQ. ID NO.:181) HIV-1RT 508-517 IYQEPFKNLK HLA-A11 188 (SEQ. ID NO.:182) HIV-1 p17 28-36KYKLKHIVW HLA-A24 188 (SEQ. ID NO.:183) HIV-1 gp120 53-62 LFCASDAKAYHLA-A24 189 (SEQ. ID NO.:184) HIV-1 gagp24 145-155 QAISPRTLNAW HLA-A25188 (SEQ. ID NO.:185) HIV-1 gagp24 167-175 EVIPMFSAL HLA-A26 188 (SEQ.ID NO.:186) HIV-1 RT 593-603 ETFYVDGAANR HLA-A26 188 (SEQ. ID NO.:187)HIV-1 gp41 775-785 RLRDLLLIVTR HLA-A31 190 (SEQ. ID NO.:188) HIV-1 RT559-568 PIQKETWETW HLA-A32 187 (SEQ. ID NO.:189) HIV-1 gp120 419-427RIKQIINMW HLA-A32 187 (SEQ. ID NO.:190) HIV-1 RT 71-79 ITLWQRPLVHLA-A*6802 188 (SEQ. ID NO.:191) HIV-1 RT 85-93 DTVLEEMNL HLA-A*6802 188(SEQ. ID NO.:192) HIV-1 RT 71-79 ITLWQRPLV HLA-A*7401 188 (SEQ. IDNO.:193) HIV-1 gag p24 148-156 SPRTLNAWV HLA-B7 188 (SEQ. ID NO.:194)HIV-1 gagp24 179-187 ATPQDLNTM HLA-B7 188 (SEQ. ID NO.:195) HIV-1 gp120303-312 RPNNNTRKSI HLA-B7 188 (SEQ. ID NO.:196) HIV-1 gp41 843-851IPRRIRQGL HLA-B7 188 (SEQ. ID NO.:197) HIV-1 p17 74-82 ELRSLYNTV HLA-B8188 (SEQ. ID NO.:198) HIV-1 nef 13-20 WPTVRERM HLA-B8 188 (SEQ. IDNO.:199) HIV-1 nef 90-97 FLKEKGGL HLA-B8 188 (SEQ. ID NO.:200) HIV-1 gagp24 183-191 DLNTMLNTV HLA-B14 191 (SEQ. ID NO.:568) HIV-1 P17 18-27KIRLRPGGKK HLA-B27 188 (SEQ. ID NO.:201) HIV-1 p17 19-27 IRLRPGGKKHLA-B27 188 (SEQ. ID NO.:202) HIV-1 gp41 791-799 GRRGWEALKY HLA-B27 188(SEQ. ID NO.:203) HIV-1 nef 73-82 QVPLRPMTYK HLA-B27 188 (SEQ. IDNO.:204) HIV-1 GP41 590-597 RYLKDQQL HLA-B27 192 (SEQ. ID NO.:205) HIV-1nef 105-114 RRQDILDLWI HLA-B*2705 188 (SEQ. ID NO.:206) HIV-1 nef134-141 RYPLTFGW HLA-B*2705 188 (SEQ. ID NO.:207) HIV-1 p17 36-44WASRELERF HLA-B35 188 (SEQ. ID NO.:208) HIV-1 GAG 262-270 TVLDVGDAYHLA-B35 188 P24 (SEQ. ID NO.:209) HIV-1 gp120 42-52 VPVWKEATTTL HLA-B35188 (SEQ. ID NO.:210) HIV-1 P17 36-44 NSSKVSQNY HLA-B35 193 (SEQ. IDNO.:221) HIV-1 gag p24 254-262 PPIPVGDIY HLA-B35 193 (SEQ. ID NO.:212)HIV-1 RT 342-350 HPDIVIYQY HLA-B35 193 (SEQ. ID NO.:213) HIV-1 gp41611-619 TAVPWNASW HLA-B35 194 (SEQ. ID NO.:214) HIV-1 gag 245-253NPVPVGNlY HLA-B35 193 (SEQ. ID NO.:215) HIV-1 nef 120-128 YFPDWQNYTHLA-B37 188 (SEQ. ID NO.:216) HIV-1 gag p24 193-201 GHQAAMQML HLA-B42188 (SEQ. ID NO.:217) HIV-1 p17 20-29 RLRPGGKKKY HLA-B42 188 (SEQ. IDNO.:218) HIV-1 RT 438-446 YPGIKVRQL HLA-B42 188 (SEQ. ID NO.:219) HIV-1RT 591-600 GAETFYVDGA HLA-B45 188 (SEQ. ID NO.:220) HIV-1 gag p24325-333 NANPDCKTI HLA-B51 188 (SEQ. ID NO.:221) HIV-1 gag p24 275-282RMYSPTSI HLA-B52 188 (SEQ. ID NO.:222) HIV-1 gp120 42-51 VPVWKEATTTHLA-B*5501 192 (SEQ. ID NO.:223) HIV-1 gag p24 147-155 ISPRTLNAW HLA-B57188 (SEQ. ID NO.:224) HIV-1 gag p24 240-249 TSTLQEQIGW HLA-B57 188 (SEQ.ID NO.:225) HIV-1 gag p24 162-172 KAFSPEVIPMF HLA-B57 188 (SEQ. IDNO.:226) HIV-1 gag p24 311-319 QASQEVKNW HLA-B57 188 (SEQ. ID NO.:227)HIV-1 gag p24 311-319 QASQDVKNW HLA-B57 188 (SEQ. ID NO.:228) HIV-1 nef116-125 HTQGYFPDWQ HLA-B57 188 (SEQ. ID NO.:229) HIV-1 nef 120-128YFPDWQNYT HLA-B57 188 (SEQ. ID NO.:230) HIV-1 gag p24 240-249 TSTLQEQIGWHLA-B58 188 (SEQ. ID NO.:231) HIV-1 p17 20-29 RLRPGGKKKY HLA-B62 188(SEQ. ID NO.:232) HIV-1 p24 268-277 LGLNKJVRMY HLA-B62 188 (SEQ. IDNO.:233) HIV-1 RT 415-426 LVGKLNWASQIY HLA-B62 188 (SEQ. ID NO.:234)HIV-1 RT 476-485 ILKEPVHGVY HLA-B62 188 (SEQ. ID NO.:235) HIV-1 nef117-127 TQGYFPDWQNY HLA-B62 188 (SEQ. ID NO.:236) HIV-1 nef 84-91AVDLSHFL HLA-B62 188 (SEQ. ID NO.:237) HIV-1 gag p24 168-175 VIPMFSALHLA-Cw*0102 188 (SEQ. ID NO.:238) HIV-1 gp120 376-384 FNCGGEFFY HLA-A29196 (SEQ. ID NO.:239) HIV-1 gp120 375-383 SFNCGGEFF HLA-B15 196 (SEQ. IDNO.:240) HIV-1 nef 136-145 PLTFGWCYKL HLA-A*0201 197 (SEQ. ID NO.:241)HIV-1 nef 180-189 VLEWRFDSRL HLA-A*0201 197 (SEQ. ID NO.:242) HIV-1 nef68-77 FPVTPQVPLR HLA-B7 197 (SEQ. ID NO.:243) HIV-1 nef 128-137TPGPGVRYPL HLA-B7 197 (SEQ. ID NO.:244) HIV-1 gag p24 308-316 QASQEVKNWHLA-Cw*0401 521 (SEQ. ID NO.:245) HIV-1 IIIB RT 273-282 VPLDEDFRKYHLA-B35 181 (SEQ. ID NO.:246) HIV-1 IIIB RT 25-33 NPDIVIYQY HLA-B35 181(SEQ. ID NO.:247) HIV-1 IIIB gp41 557-565 RAIEAQAHL HLA-B51 181 (SEQ. IDNO.:248) HIV-1 IIIB RT 231-238 TAFTIPSI HLA-B51 181 (SEQ. ID NO.:249)HIV-I IIIB p24 215-223 VHPVHAGPIA HLA-B*5501 181 (SEQ. ID NO.:250) HIV-1IIIB gp120 156-165 NCSFNISTSI HLA-Cw8 181 (SEQ. ID NO.:251) HIV-I IIIBgp120 241-249 CTNVSTVQC HLA-Cw8 181 (SEQ. ID NO.:252) HIV-1 5F2 gp120312-320 IGPGRAFHT H2-Dd 198 (SEQ. ID NO.:253) HIV-1 5F2 pol 25-33NPDIVIYQY HLA-B*3501 199 (SEQ. ID NO.:254) HIV-15F2 pol 432-441EPIVGAETFY HLA-B*3501 199 (SEQ. ID NO.:255) HIV-1 5F2 pol 432-440EPIVGAETF HLA-B*3501 199 (SEQ. ID NO.:256) HIV-1 5F2 pol  6-14 SPAIFQSSMHLA-B*3501 199 (SEQ. ID NO.:257) HIV-1 5F2 pol 59-68 VPLDKDFRKYHLA-B*3501 199 (SEQ. ID NO.:258) HIV-1 5F2 pol  6-14 IPLTEEAELHLA-B*3501 199 (SEQ. ID NO.:259) HIV-1 5F2 nef 69-79 RPQVPLRPMTYHLA-B*3501 199 (SEQ. ID NO.:260) HIV-1 5F2 nef 66-74 FPVRPQVPLHLA-B*3501 199 (SEQ. ID NO.:261) HIV-1 5F2 env 10-18 DPNPQEVVLHLA-B*3501 199 (SEQ. ID NO.:262) HIV-1 5F2 env  7-15 RPIVSTQLLHLA-B*3501 199 (SEQ. ID NO.:263) HIV-1 5F2 pol  6-14 IPLTEEAEL HLA-B51199 (SEQ. ID NO.:264) HIV-1 5F2 env 10-18 DPNPQEVVL HLA-B51 199 (SEQ. IDNO.:265) HIV-1 5F2 gagp24 199-207 AMQMLKETI H2-Kd 198 (SEQ. ID NO.:266)HIV-2 gagp24 182-190 TPYDrNQML HLA-B*5301 200 (SEQ. ID NO.:267) HIV-2gag 260-269 RRWIQLGLQKV HLA-B*2703 188 (SEQ. ID NO.:268) HIV-1 5F2 gp41593-607 GIWGCSGKLICTTA HLA-B17 201 V (SEQ. ID NO.:269) HIV-1 5F2 gp41753-767 ALIWEDLRSLCLFS HLA-B22 201 Y (SEQ. ID NO.:270) HPV 6b E7 21-30GLHCYEQLV HLA-A*0201 202 (SEQ. ID NO.:271) HPV 6b E7 47-55 PLKQHFQIVHLA-A*0201 202 (SEQ. ID NO.:272) HPV11 E7  4-12 RLVTLKDIV HLA-A*0201 202(SEQ. ID NO.:273) HPV16 E7 86-94 TLGIVCPIC HLA-A*0201 129 (SEQ. IDNO.:274) HPV16 E7 85-93 GTLGIVCPI HLA-A*0201 129 (SEQ. ID NO.:275) HPV16E7 12-20 MLDLQPETT HLA-A*0201 129 (SEQ. ID NO.:276) HPV16 E7 11-20YMLDLQPETT HLA-A*0201 203 (SEQ. ID NO.:277) HPV16 E6 15-22 RPRKLPQLHLA-B7 204 (SEQ. ID NO.:278) HPV16 E6 49-57 RAHYNIVTF HW-Db 205 (SEQ. IDNO.:279) HSV gp B 498-505 SSIEFARL H2-Kb 206 (SEQ. ID NO.:280) HSV-1 gpC 480-488 GIGIGVLAA HLA-A*0201 104 (SEQ. ID NO.:281) HSV-1 ICP27 448-456DYATLGVGV H2-Kd 207 (SEQ. ID NO.:282) HSV-1 ICP27 322-332 LYRTFAGNPRAH2-Kd 207 (SEQ. ID NO.:283) HSV-1 UL39 822-829 QTFDFGRL H2-Kb 208 (SEQ.ID NO.:284) HSV-2 gpC 446-454 GAGIGVAVL HLA-A*0201 104 (SEQ. ID NO.:285)HLTV-1 TAX 11-19 LLFGYPVYV HLA-A*0201 209 (SEQ. ID NO.:286) Influenza MP58-66 GILGFVFTL HLA-A*0201 68, 169, (SEQ. ID NO.:287) 209, 210, 211Influenza MP 59-68 ILGFVFTLTV HLA-A*0201 168, 212, (SEQ. ID NO.:288) 213Influenza NP 265-273 ILRGSVAHK HLA-A3 214 (SEQ. ID NO.:289) Influenza NP91-99 KTGGPIYKR HLA-A*6801 215, 216 (SEQ. ID NO.:290) Influenza NP380-388 ELRSRYWAI HLA-B8 217 (SEQ. ID NO.:291) Influenza NP 381-388LRSRYWAI HLA-B*2702 218 (SEQ. ID NO.:292) Influenza NP 339-347 EDLRVLSFIHLA-B*3701 219 (SEQ. ID NO.:293) Influenza NSI 158-166 GEISPLPSL HLA-B44220 (SEQ. ID NO.:294) Influenza NP 338-346 FEDLRVLSF HLA-B44 220 (SEQ.ID NO.:295) Influenza NSI 158-166 GEISPLPSL HLA-B*4402 220 (SEQ. IDNO.:294) Influenza NP 338-346 FEDLRVLSF HLA-B*4402 220 (SEQ. ID NO.:295)Influenza PBI 591-599 VSDGGPKLY HLA-A1 214, 29 (SEQ. ID NO.:296)Influenza A NP 44-52 CTELKLSDY HLA-A1 29 (SEQ. ID NO.:297) Influenza NSI122-130 AIMDKNIIL HLA-A*0201 221 (SEQ. ID NO.:298) Influenza A NSI123-132 IMDKNIILKA HLA-A*0201 221 (SEQ. ID NO.:299) Influenza A NP383-391 SRYWAIRTR HLA-B*2705 160, 184 (SEQ. ID NO.:300) Influenza A NP147-155 TYQRTRALV H2-Kd 222, 223 (SEQ. ID NO.:301) Influenza A HA210-219 TYVSVSTSTL H2-Kd 224, 225 (SEQ. ID NO. 302) Influenza A HA518-526 IYSTVASSL H2-Kd 224 (SEQ. ID NO. 303) Influenza A HA 259-266FEANGNLI H2-Kk 226, 227, (SEQ. ID NO.:304) 228 Influenza A HA 10-18IEGGWTGMI H2-Kk 226, 227, (SEQ. ID NO.:305) 228 Influenza A NP 50-57SDYEGRLI H2-Kk 229, 230 (SEQ. ID NO.. 306) Influenza a NSI 152-160EEGAIVGEI H2-Kk 231 (SEQ. ID NO.:307) Influenza NP 336-374 ASNENMETMH2Db 168, 222, A34 (SEQ. ID NO.:308) 219 Influenza NP 366-374 ASNENMDAMH2Db 232 A68 (SEQ. ID NO.:309) Influenza B NP 85-94 KLGEFYNQMMHLA-A*0201 233 (SEQ. ID NO.:310) Influenza B NP 85-94 KAGEFYNQMMHLA-A*0201 234 (SEQ. ID NO.:311) Influenza HA 204-212 LYQNVGTYV H2Kd 235JAP (SEQ. ID NO.312) Influenza HA 210-219 TYVSVGTSTL H2-Kd 225 JAP (SEQ.ID NO.:313) Influenza HA 523-531 VYQILATYA H2-Kd 235 JAP (SEQ. IDNO.314) Influenza HA 529-537 IYATVAGSL H2-Kd 235 JAP (SEQ. ID NO.315)Influenza HA 210-219 TYVSVGTSTI H2-Kd 236 JAP (L > I) (SEQ. ID NO.:316)Influenza HA 255-262 FESTGNLI H2-Kk 237 JAP (SEQ. ID NO.:317) JHMV cAg318-326 APTAGAFFF H2-Ld 238 (SEQ. ID NO.:318) LCMV NP 118-126 RPQASGVYMH2-Ld 239-240 (SEQ. ID NO.319) LCMV NP 396-404 FQPQNGQFI H2-Db 241 (SEQ.ID NO.320) LCMV GP 276-286 SGVENPGGYCL H2-Db 242 (SEQ. ID NO.:321) LCMVGP 33-42 KAVYNFATCG H2-Db 243, 244 (SEQ. ID NO.:322) MCMV pp89 168-176YPHFMPTNL H2-Ld 245 (SEQ. ID NO.323) MHV spike 510-518 CLSWNGPHL H2-Db248 protein (SEQ. ID NO.324) MMTV env gp 474-482 SFAVATTAL H2-Kd 246 36(SEQ. ID NO.:325) MMTV gag p27 425-433 SYETFISRL H2-Kd 246 (SEQ. IDNO.:326) MMTV env gp73 544-551 ANYDFICV H2-Kb 247 (SEQ. ID NO.:327) MuLVenv p15E 574-581 KSPWFTTL H2-Kb 249, 250 (SEQ. ID NO.:328) MuLV env gp70189-196 SSWDFITV H2-Kb 251, Sijts (SEQ. ID NO.:329) et al. SubmittedMuLV gag 75K 75-83 CCLCLTVFL H2-Db 252 (SEQ. ID NO.:330) MuLV env gp70423-431 SPSYVYHQF H2Ld 253 (SEQ. ID NO.:331) MV F protein 437-447SRRYPDAVYLH HLA-B*2705 254 (SEQ. ID NO.:332) Mv F protein 438-446RRYPDAVYL HLA-B*2705 255 (SEQ. ID NO.333) Mv NP 281-289 YPALGLHEF H2-Ld256 (SEQ. ID NO.:334) Mv HA 343-351 DPVIDRLYL H2-Ld 257 (SEQ. ID NO.335)MV HA 544-552 SPGRSFSYF H2-Ld 257 (SEQ. ID NO.:336) Poliovirus VP1111-118 TYKDTVQL H2-kd 258 (SEQ. ID NO.:337) Poliovirus VP1 208-217FYDGFSKVPL H2-Kd 258 (SEQ. ID NO.:338) Pseudorabies G111 455-463IAGIGILAI HLA-A*0201 104 virus gp (SEQ. ID NO.:339) Rabiesvirus NS197-205 VEAEIAHQI H2-Kk 227-227 (SEQ. ID NO.:340) Rotavirus VP7 33-40llYRFLLl H2-Kb 259 (SEQ. ID NO.:341) Rotavirus VP6 376-384 VGPVFPPGMH2-Kb 260 (SEQ. ID NO.:342) Rotavirus VP3 585-593 YSGYIFRDL H2-Kb 260(SEQ. ID NO.:343) RSV M2 82-90 SYIGSINNI H2-Kd 261 (SEQ. ID NO.:344) SIVgagp11C 179-190 EGCTPYDTNQML Mamu-A*01 266 (SEQ. ID NO.:345) SV NP324-332 FAPGNYPAL H2-Db 262 (SEQ. ID NO.:346) SV NP 324-332 FAPCTNYPALH2-Kb 263, 264, (SEQ. ID NO.:346) 265 SV40 T 404-411 VVYDFLKC H2-Kb 267(SEQ. ID NO.:347) SV40 T 206-215 SAINNYAQKL H2-Db 268, 269 (SEQ. IDNO.:348) SV40 T 223-231 CKGVNKEYL H2-Db 268, 269 (SEQ. ID NO.349) SV40 T489-497 QGINNLDNL H2-Db 268, 269 (SEQ. ID NO.:350) SV40 T 492-500NNLDNLRDY(L) H2-Db 270 (501) (SEQ. ID NO.:351) SV40 T 560-568 SEFLLEKRIH2-Kk 271 (SEQ. ID NO.352) VSV NP 52-59 RGYVYQGL H2-Kb 272 (SEQ. IDNO.:353)

Table II sets forth antigens identified from various protein sources.The Table is extracted from Table 4.2 in the Rammensee book with thereferences in Table H being the same as the references in the RammenseeTable 4.2.

TABLE II HLA Class I Motifs HLA-A1 Position (Antigen) Source Ref. T cellEADPTGHSY MAGE-1 161-169 27, 28 epitopes (SEQ. ID NO.:354) VSDGGPNLYInfluenza A PB 21, 23 (SEQ. ID NO.:355) 1591-599 CTELKLSDY Influenza ANP 23 (SEQ. ID NO.:356) 44-52 EVDPIGHLY MAGE-3 168-176 29, 30 (SEQ. IDNO.:357) HLA-A201 MLLSVPLLLG Calreticulin signal 34, 35, (SEQ. IDNO.:358) sequence 1-10 36, 37 STBXQSGXQ HBV PRE-S PROTEIN 43 (SEQ. IDNO.:359) 141-149 YMDGTMSQV Tyrosinase 369-377 45 (SEQ. ID NO.:360)ILKEPVHGV HIV-I RT 476-484 4, 31, (SEQ. ID NO.:361) 47 LLGFVFTLTVInfluenza MP 59-68 4, 39 (SEQ. ID NO.:362) LLFGYPVYVV HTLV-1 tax 11-1940 (SEQ. ID NO.:363) GLSPTVWLSV HBV sAg 348-357 48 (SEQ. ID NO.:364)WLSLLVPFV HBV sAg 335-343 49, 50. (SEQ. ID NO.:365) 51 FLPSDFFPSV HBVcAg 18-27 52 (SEQ. ID NO.:366) CLG0LLTMV EBV LMP-2 426-434 48 (SEQ. IDNO.:367) FLAGNSAYEYV HCMV gp 618-628B 53 (SEQ. ID NO.:368) KLGEFYNQMMInfluenza BNP 85-94 54 (SEQ. ID NO.:369) KLVALGINAV HCV-1 NS3 400-409 55(SEQ. ID NO.:370) DLMGYIPLV HCV MP 17-25 56 (SEQ. ID NO.:371) RLVTLKDIVHPV 11 EZ 4-12 34, 35 (SEQ. ID NO.:372) MLLAVLYCL Tyrosinase 1-9 57, 58,(SEQ. ID NO-373) 59, 68 AAGIGILTV Melan A\Mart-127-35 60 (SEQ. IDNO.:374) YLEPGPVTA Pmel 17/gp 100 61 (SEQ. ID NO.:375) 480-488ILDGTATLRL Pmel 17/gp 100 62 (SEQ. ID NO.:376) 457-466 LLDGTATLRL PmelgplOO 457-466 62 (SEQ. ID NO.:377) ITDQVPFSV Pmel gp 100 209-217 62(SEQ. ID NO.:378) KTWGQYWQV Pmel gp 100 154-162 62 (SEQ. ID NO.:379)TITDQVPFSV Pmel gp 100 208-217 62 (SEQ. ID NO.:380) AFHIIVAREL HIV-I nef190-198 63 (SEQ. ID NO.:381) YLNKIQNSL P. falciparum CSP 64 (SEQ. IDNO.:382) 334-342 MMRKLAILSV P. falciparum CSP 64 (SEQ. ID NO.:383) 1-10KAGEFYNQMM Influenza BNP 85-94 65 (SEQ. ID NO.:384) NIAEGLRAL EBNA-1480-488 66 (SEQ. ID NO.:385) NLRRGTALA EBNA-1 519-527 66 (SEQ. IDNO.:386) ALAIPQCRL EBNA-1 525-533 66 (SEQ. ID NO.:387) VLKDAIKDL EBNA-1575-582 66 (SEQ. ID NO.:388) FMVFLQTHI EBNA-1 562-570 66 (SEQ. IDNO.:389) HLIVDTDSL EBNA-2 15-23 66 (SEQ. ID NO.:390) SLGNPSLSV EBNA-222-30 66 (SEQ. ID NO.391) PLASAMRML EBNA-2 126-134 66 (SEQ. ID NO.392)RMLWMANYI EBNA-2 132-140 66 (SEQ. ID NO.:393) MLWMANYIV EBNA-2 133-14166 (SEQ. ID NO.:394) ILPQGPQTA EBNA-2 151-159 66 (SEQ. ID NO.:395)PLRPTAPTTI EBNA-2 171-179 66 (SEQ. ID NO.:396) PLPPATLTV EBNA-2 205-21366 (SEQ. ID NO.:397) RMHLPVLHV EBNA-2 246-254 66 (SEQ. ID NO.:398)PMPLPPSQL EBNA-2 287-295 66 (SEQ. ID NO.:399) QLPPPAAPA EBNA-2 294-30266 (SEQ. ID NO.:400) SMPELSPVL EBNA-2 381-389 66 (SEQ. ID NO.:401)DLDESWDYI EBNA-2 453-461 66 (SEQ. ID NO.:402) PLPCVLWPVV BZLFl 43-51 66(SEQ. ID NO.:403) SLEECDSEL BZLFl 167-175 66 (SEQ. ID NO.:404) EIKRYKNRVBZLFI 176-184 66 (SEQ. ID NO.:405) QLLQFIYREV BZLFl 195-203 66 (SEQ. IDNO.:406) LLQHYREVA BZLFI 196-204 66 (SEQ. ID NO.:407) LLKQMCPSL BZLFI217-225 66 (SEQ. ID NO.:408) SIIPRTPDV BZLFI 229-237 66 (SEQ. IDNO.:409) AIMDKNIIL Influenza A NS1 67 (SEQ. ID NO.:410) 122-130IMDKNIILKA Influenza A NS1 67 (SEQ. ID NO.411) 123-132 LLALLSCLTV HCV MP63-72 69 (SEQ. ID NO.:412) ILHTPGCV HCV MP 105-112 69 (SEQ. ID NO.:413)QLRRHIDLLV HCV env E 66-75 69 (SEQ. ID NO.:414) DLCGSVFLV HCV env E88-96 69 (SEQ. ID NO.:415) SMVGNWAKV HCV env E 172-180 69 (SEQ. IDNO.:416) HLHQNIVDV HCV NSI 308-316 69 (SEQ. ID NO.:417) FLLLADARV HCVNSI 340-348 69 (SEQ. ID NO.:418) GLRDLAVAVEPVV HCV NS2 234-246 69 (SEQ.ID NO.:419) SLLAPGAKQNV HCV NS1 18-28 69 (SEQ. ID NO.:420) LLAPGAKQNVHCV NS1 19-28 69 (SEQ. ID NO.:421) FLLSLGIHL HBV pol 575-583 70 (SEQ. IDNO.:422) SLYADSPSV HBV pol 816-824 70 (SEQ. ID NO.:423) GLSRYVARL HBVPOL 455-463 70 (SEQ. ID NO.:424) KIFGSLAFL HER-2 369-377 71 (SEQ. IDNO.:425) ELVSEFSRM HER-2 971-979 71 (SEQ. ID NO.:426) KLTPLCVTL HIV-I gp160 72 (SEQ. ID NO.:427) 120-128 SLLNATDIAV HIV-I GP 160 72 (SEQ. IDNO.:428) 814-823 VLYRYGSFSV Pmel gp100 476-485 62 (SEQ. ID NO.:429)YIGEVLVSV Non-filament 73 (SEQ. ID NO.:430) forming class I myosinfamily (HA-2)** LLFNILGGWV HCV NS4 192-201 74 (SEQ. ID NO.:431)LLVPFVQWFW HBV env 338-347 74 (SEQ. ID NO.:432) ALMPLYACI HBV pol642-650 74 (SEQ. ID NO.:433) YLVAYQATV HCV NS3 579-587 74 (SEQ. IDNO.:434) TLGIVCPIC HIPV 16 E7 86-94 74 (SEQ. ID NO.:435) YLLPRRGPRL HCVcore protein 74 (SEQ. ID NO.:436) 34-43 LLPIFFCLWV HBV env 378-387 74(SEQ. ID NO.:437) YMDDVVLGA HBV Pol 538-546 74 (SEQ. ID NO.:438)GTLGIVCPI HPV16 E7 85-93 74 (SEQ. ID NO.:439) LLALLSCLTI HCV MP 63-72 74(SEQ. ID NO.:440) MLDLQPETT HPV 16 E7 12-20 74 (SEQ. ID NO.:441)SLMAFTAAV HCV NS4 174-182 75 (SEQ. ID NO.:442) CINGVCWTV HCV NS3 67-7575 (SEQ. ID NO.:443) VMNILLQYVV Glutamic acid 76 (SEQ. ID NO.:444)decarboxylase 114-123 ILTVILGVL Melan A/Mart-32-40 77 (SEQ. ID NO.:445)FLWGPRALV MAGE-3 271-279 78 (SEQ. ID NO.:446) LLCPAGHAV HCV NS3 163-17154 (SEQ. ID NO.:447) ILDSFDPLV HCV NSS 239-247 54 (SEQ. ID NO.:448)LLLCLIFLL HBV env 250-258 79 (SEQ. ID NO.:449) LIDYQGMLPV HBV env260-269 79 (SEQ. ID NO.:450) SIVSPFIPLL HBV env 370-379 79 (SEQ. IDNO.:451) FLLTRILTI HBV env 183-191 80 (SEQ. ID NO.:452) HLGNVKYLV P.faciparum TRAP 81 (SEQ. ID NO.:453) 3-11 GIAGGLALL P. faciparum TRAP 81(SEQ. ID NO.:454) 500-508 ILAGYGAGV HCV NS S4A 236-244 82 (SEQ. IDNO.:455) GLQDCTMLV HCV NS5 714-722 82 (SEQ. ID NO.:456) TGAPVTYSTY HCVNS3 281-290 83 (SEQ. ID NO.:457) VIYQYMDDLV HIV-1RT 179-187 84 (SEQ. IDNO.:458) VLPDVFIRCV N-acetylgluco 85 saminyltransferase (SEQ. IDNO.:459) V Gnt-V intron VLPDVFIRC N-acetylgluco 85 (SEQ. ID NO.:460)saminyltransferase V Gnt-V intron AVGIGIAVV Human CD9 86 (SEQ. IDNO.:461) LVVLGLLAV Human 86 (SEQ. ID NO.:462) glutamyltransferaseALGLGLLPV Human G protein 86 (SEQ. ID NO.:463) coupled receptor 164-172GIGIGVLAA HSV-I gp C 480-488 86 (SEQ. ID NO.:281) GAGIGVAVL HSV-2 gp C446-454 86 (SEQ. ID NO.:464) IAGIGILAI Pseudorabies gpGIN 86 (SEQ. IDNO.:465) 455-463 LIVIGILIL Adenovirus 3 E3 9kD 86 (SEQ. ID NO.:466)30-38 LAGIGLIAA S. Lincolnensis 86 (SEQ. ID NO.:467) ImrA VDGIGILTIYeast ysa-1 77-85 86 (SEQ. ID NO.:468) GAGIGVLTA B. polymyxa, 86 (SEQ.ID NO.:469) βcndoxylanase 149-157 AAGIGIIQI E. coli methionine 86 (SEQ.ID NO.:470) synthase 590-598 QAGIGILLA E. coli 86 (SEQ. ID NO.:471)hypothetical protein 4-12 KARDPHSGHFV CDK4w1 22.32 87 (SEQ. ID NO.:472)KACDPI-ISGIIFV CDK4-R24C 22-32 87 (SEQ. ID NO.:473) ACDPFISGHFVCDK4-R24C 23-32 87 (SEQ. ID NO.:474) SLYNTVATL HIV-I gag p 17 99 (SEQ.ID NO.:475) 77-85 ELVSEFSRV HER-2, m > V 89 (SEQ. ID NO.:476)substituted 971-979 RGPGRAFVTI HIV-I gp 160 90 (SEQ. ID NO.:477) 315-329HMWNFISGI HCV NS4A 91 (SEQ. ID NO.:478) 149-157 NLVPMVATVQ HCMV pp65495-504 92 (SEQ. ID NO.:479) GLHCYEQLV HPV 6b E7 21-30 93 (SEQ. IDNO.:480) PLKQHFQIV HPV 6b E7 47-55 93 (SEQ. ID NO.:481) LLDFVRFMGVEBNA-6 284-293 95 (SEQ. ID NO.:482) AIMEKNIML Influenza Alaska 67 (SEQ.ID NO.:483) NS 1 122-130 YLKTIQNSL P. falciparum cp36 96 (SEQ. IDNO.:484) CSP YLNKIQNSL P. falciparurn cp39 96 (SEQ. ID NO.:485) CSPYMLDLQPETT HPV 16 E7 11-20* 97 (SEQ. ID NO.:486) LLMGTLGIV HPV16 E782-90** 97 (SEQ. ID NO.:487) TLGIVCPI HPV 16 E7 86-93 97 (SEQ. IDNO.:488) TLTSCNTSV HIV-1 gp120 197-205 98 (SEQ. ID NO.:489) KLPQLCTELHPV 16 E6 18-26 97 (SEQ. ID NO.:490) TIHDIILEC HPV16 E6 29-37 97 (SEQ.ID NO.:491) LGIVCPICS HPV16 E7 87-95 97 (SEQ. ID NO.:492) VILGVLLLIMelan A/Mart-1 68 (SEQ. ID NO.:493) 35-43 ALMDKSLHV Melan A/Mart-1 68(SEQ. ID NO.:494) 56-64 GILTVILGV Melan A/Mart-1 68 (SEQ. ID NO.:495)31-39 T cell MINAYLDKL P. Falciparum 81 epitopes (SEQ. ID NO.:496) STARP523-531 AAGIGILTV Melan A/Mart- 100 (SEQ. ID NO.:497) 127-35 FLPSDFFPSVHBV cAg 18-27 51 (SEQ. ID NO.:498) Motif SVRDRLARL EBNA-3 464-472 101unknown T cell (SEQ. ID NO.:499) epitopes T cell AAGIGILTV MelanA/Mart-1 100 epitopes (SEQ. ID NO.:497) 27-35 FAYDGKDYI Human MHC I-ot99 (SEQ. ID NO.:500) 140-148 T cell AAGIGILTV Melan A/Mart-1 100epitopes (SEQ. ID NO.:497) 27-35 FLPSDFFPSV HBV cAg 18-27 51 (SEQ. IDNO.:498) Motif AAGIGILTV Meland A/Mart-1 100 unknown 27-35 T cell (SEQ.ID NO.:497) epitopes FLPSDFFPSV HBV cAg 18-27 51 (SEQ. ID NO.:498)AAGIGILTV Melan A/Mart-1 100 (SEQ. ID NO.:497) 27-35 ALLAVGATK Pme117 gp100 17-25 107 (SEQ. ID NO.:501) T cell RLRDLLLIVTR HIV-1 gp41 768-778108 epitopes (SEQ. ID NO.:502) QVPLRPMTYK HIV-1 nef 73-82 109 (SEQ. IDNO.:503) TVYYGVPVWK HIV-1 gp120-36-45 110 (SEQ. ID NO.:504) RLRPGGKKKHIV-1 gag p 17 110 (SEQ. ID NO.:505) 20-29 ILRGSVAHK Influenza NP 21(SEQ. ID NO.:506) 265-273 RLRAEAGVK EBNA-3 603-611 111 (SEQ. ID NO.:507)RLRDLLLIVTR HIV-1 gp41 112 (SEQ. ID NO.:502) 770-780 VYYGVPVWK HIV-I GP113 (SEQ. ID NO.:508) 120 38-46 RVCEKMALY HCV NS5 575-583 114 (SEQ. IDNO.:509) Motif KIFSEVTLK Unknown; muta Wolfel unknown melanoma peptideet al., ted (p I 83L) pers. 175-183 Comm. T cell (SEQ. ID NO.:510)epitope YVNVNMGLK* HBV cAg 88-96 116 (SEQ. ID NO.:511) T cell IVTDFSVIKEBNA-4 416-424 115, 117 epitopes (SEQ. ID NO.:512) ELNEALELK P53 343-351115 (SEQ. ID NO.:513) VPLRPMTYK HIV-1 NEF 74-82 115 (SEQ. ID NO.:514)AIFQSSMTK HIV-I gag p24 115 (SEQ. ID NO.:515) 325-333 QVPLRPMTYK HIV-1nef 73-82 118 (SEQ. ID NO.:516) TINYTIFK HCV NSI 238-246 114 (SEQ. IDNO.:517) AAVDLSHFLKEK HIV-1 nef 83-94 120 (SEQ. ID NO.:518) ACQGVGGPGGHKHIV-1 I I 1B p24 122 (SEQ. ID NO.:519) 349-359 HLA-A24 SYLDSGIHF*β-catenin, mutated 123 (proto-onocogen) 29-37 (SEQ. ID NO.:520) T cellRYLKDQQLL HIV GP 41 583-591 124 epitopes (SEQ. ID NO.:521) AYGLDFYIL P15melanoma Ag 125 (SEQ. ID NO.:522) 10-18 AFLPWHRLFL Tyrosinase 206-215126 (SEQ. ID NO.:523) AFLPWHRLF Tyrosinase 206-214 126 (SEQ. ID NO.:524)RYSIFFDY Ebna-3 246-253 101 (SEQ. ID NO.:525) T cell ETINEEAAEW HIV-1gag p24 127 epitope (SEQ. ID NO.:526) 203-212 T cell STLPETTVVRR HBV cAg141-151 129 epitopes (SEQ. ID NO.:527) MSLQRQFLR ORF 3P-gp75 130 (SEQ.ID NO.:528) 294-321 (bp) LLPGGRPYR TRP (tyrosinase 131 (SEQ. ID NO.:529)rel.) 197-205 T cell IVGLNKIVR HIV gag p24 132, 133 epitope (SEQ. IDNO.:530) 267-267-275 AAGIGILTV Melan A/Mart-127 35 100 (SEQ. ID NO.:531)

Table III sets forth additional antigens useful in the invention thatare available from the Ludwig Cancer Institute. The Table refers topatents in which the identified antigens can be found and as such areincorporated herein by reference. TRA refers to the tumor-relatedantigen and the LUD No. refers to the Ludwig Institute number.

TABLE III LUD Patent Date Patent Peptide TRA No. No. Issued (Antigen)HLA MAGE-4 5293 5,405,940 11 Apr. 1995 EVDPASNTY HLA-A1 (SEQ. IDNO.:532) MAGE-41 5293 5,405,940 11 Apr. 1995 EVDPTSNTY HLA-A I (SEQ IDNO:533) MAGE-5 5293 5,405,940 11 Apr. 1995 EADPTSNTY HLA-A I (SEQ IDNO:534) MAGE-51 5293 5,405,940 11 Apr. 1995 EADPTSNTY HLA-A I (SEQ IDNO:534) MAGE-6 5294 5,405,940 11 Apr. 1995 EVDPIGHVY HLA-A1 (SEQ IDNO:535) 5299.2 5,487,974 30 Jan. 1996 MLLAVLYCLL HLA-A2 (SEQ ID NO:536)5360 5,530,096 25 Jun. 1996 MLLAVLYCL HLA-B44 (SEQ ID NO:537) Tyrosinase5360.1 5,519,117 21 May 1996 SEIWRDIDFA HLA-B44 (SEQ ID NO:538)SEIWRDIDF (SEQ ID NO:539) Tyrosinase 5431 5,774,316 28 Apr. 1998XEIWRDIDF HLA-B44 (SEQ ID NO:540) MAGE-2 5340 5,554,724 10 Sep. 1996STLVEVTLGEV HLA-A2 (SEQ ID NO:541) LVEVTLGEV (SEQ ID NO:542) VIFSKASEYL(SEQ ID NO:543) IIVLAIIAI (SEQ ID NO:544) KIWEELSMLEV (SEQ ID NO:545)LIETSYVKV (SEQ ID NO:546) 5327 5,585,461 17 Dec. 1996 FLWGPRALV HLA-A2(SEQ ID NO:547) TLVEVTLGEV (SEQ ID NO:548) ALVETSYVKV (SEQ ID NO:549)MAGE-3 5344 5,554,506 10 Sep. 1996 KIWEELSVL HLA-A2 (SEQ ID NO:550)MAGE-3 5393 5,405,940 11 Apr. 1995 EVDPIGHLY HLA-A1 (SEQ ID NO:551) MAGE5293 5,405,940 11 Apr. 1995 EXDX5Y HLA-A1 (SEQ. ID NO.:552) (but notEADPTGHSY) (SEQ. ID NO.553) E (A/V) D X5 Y (SEQ. ID NO.:554) E (A/V) D PX4 Y (SEQ. ID NO.:555) E (A/V) D P (I/A/T) X3 Y (SEQ. ID NO.:556) E(A/V) D P (I/A/T) (G/S) X2 Y (SEQ. ID NO.:557) E (A/V) D P (I/A/T) (G/S)(H/N) X Y E (A/V) DP (I/A/T) (G/S) (H/N) (L/T/V) Y (SEQ. ID NO.:559)MAGE-1 5361 5,558.995 24 Sep. 1996 ELHSAYGEPRKLLTQD HLA-C (SEQ IDNO:560) Clone 10 EHSAYGEPRKLL (SEQ ID NO:561) SAYGEPRKL (SEQ ID NO:562)MAGE-1 5253.4 TBA TBA EADPTGHSY HLA-A I (SEQ ID NO:563) BAGE 5310.1 TBATBA MAARAVFLALSAQLLQARLMKE HLA-C (SEQ ID NO:564) Clone 10MAARAVFLALSAQLLQ HLA-C (SEQ ID NO:565) Clone 10 AARAVFLAL HLA-C (SEQ IDNO:566) Clone 10 GAGE 5323.2 5,648,226 15 Jul. 1997 YRPRPRRY HLA- CW6(SEQ. ID NO.:567)

Preferred peptide antigens are those of tumor associated antigens (TAA)and chronic infections. Particularly preferred peptide antigens arederived from tyrosinose, gp100 or Melan A for the treatment of melanoma.

The peptide antigens of this invention are readily prepared usingstandard peptide synthesis means known in the art. Generally they can beprepared commercially by one of numerous companies that do chemicalsynthesis. An example is American Peptides, Inc., where the distributoris CLINALFA AG (Laufelfingen, Switzerland). The antigens are prepared inaccordance with GMP standards. Purity is assessed by analytical HPLC.The product is characterized by amino-acid analysis and tested forsterility and the absence of pyrogens.

In delivering an appropriate antigen, e.g., a polypeptide, to theanimal's system it may be delivered directly as the polypeptide, or itmay be delivered indirectly, e.g., using a DNA construct or vector, or arecombinant virus that codes for the desired antigen. Any vector drivingexpression in a professional antigen presenting cell is suitable forthis purpose. In the indirect delivery, the antigen is expressed in thecell, to be presented by the MHC Class I on the surface of the cell tostimulate the CTL response.

In a preferred embodiment of the invention an encoded antigen can bedelivered in the form of a naked plasmid expression vector. Particularlyuseful constructs are disclosed in U.S. patent application Ser. No.09/561,572 entitled EXPRESSION VECTORS ENCODING EPITOPES OFTARGET-ASSOCIATED ANTIGENS which is incorporated herein by reference inits entirety. The feasibility of and general procedures related to theuse of naked DNA for immunization are described in U.S. Pat. No.5,589,466, entitled “INDUCTION OF A PROTECTIVE IMMUNE RESPONSE IN AMAMMAL BY INJECTING A DNA SEQUENCE” and U.S. Pat. No. 5,679,647,entitled “METHODS AND DEVICES FOR IMMUNIZING A HOST AGAINSTTUMOR-ASSOCIATED ANTIGENS THROUGH ADMINISTRATIONS OF NAKEDPOLYNUCLEOTIDES WHICH ENCODE TUMOR-ASSOCIATED ANTIGENIC PEPTIDES” whichare herein incorporated by reference in their entirety. However theformer teaches only intramuscular or intradermal injection while thelatter teaches only administration to skin or mucosa. Administrationdirectly to the lymphatic system is greatly more efficient (see examples6-9, below). Single bolus injection into lymph node required only 0.1%of the dose required in order to obtain a similar level of CTL responseby intramuscular (i.m.) injection. It is therefore feasible to establisha protective response against systemic viral infection with a singlebolus delivered i.ln., but not with a dose nearing the practical limitdelivered i.m. Even repeated bolus injections i.m. failed to establish aprotective response against a peripheral virus infection or transplantedtumor whereas lower doses administered i.m. were completely effective.

In another embodiment of the invention an encoded antigen can bedelivered in the form of a viral vector. A wide array of viruses withmodified genomes adapted to express interposed reading frames but oftenno, or at least a reduced number of, viral proteins are known in theart, including without limitation, retroviruses including lentiviruses,adenoviruses, parvoviruses including adeno-associated virus,herpesviruses, and poxviruses including vaccinia virus. Such viralvectors facilitate delivery of the nucleic acid component into the cellallowing for expression. A subset of these vectors, such as retrovirusesand parvoviruses, also promotes integration of their nucleic acidcomponent into the host genome, whereas others do not.

Bacteria can also serve as vectors, analogously to viruses, i.e. theycan be used to deliver a nucleic acid molecule capable of causingexpression of an antigen. For example, a strain of Listeriamonocytogenes has been devised that effects its own lysis upon enteringthe cytosol of macrophages (its normal target), thereby releasingplasmid from which antigen was subsequently expressed (Dietrich, G. etal. Biotechnology 16:181-185, 1998 which is herein incorporated byreference in their entirety). Shigela flexneri and Escherichia coli havebeen similarly used (Sizemore, D. R. et al. Science 270:299-302, 1995,and Courvalin, P. et al. Life Sci. 318:1207-1212, 1995, respectively,which are herein incorporated by reference in their entirety).

The use of microbial vectors for nucleic acid delivery can becomplicated by the immune reactions the vectors themselves provoke. Whenprolonged or repeated administration is required, antibody elicited bythe earlier treatment can prevent useful quantities of the vector fromever reaching its intended host. However, by direct administration into,for example, a lymph node, the combination of proximity to host cellsand the much reduced effective dose makes it possible to administer adose capable of evading or overwhelming an existing antibody titer.

The word vector has been used, here and elsewhere, in reference toseveral modalities and variously modified (e.g. expression vector, viralvector, delivery vector, etc.). The underlying principle here is that anucleic acid capable of causing expression of an antigen ultimatelyarrives in an APC, rather than the antigen itself. Unless modified,explicitly or by local context, wherever the term vector is used herein,it is intended to encompass all such possibilities.

These foregoing techniques are distinct from the approach of modifyingthe microbial genome (including extra-chromosomal DNA) so that theantigen is produced as a component of the microbe (virus, bacteria,fungi, protazoan, etc., etc.), which is then itself administered as theimmunogen. Obviously, the genomic modification would most likely involvethe use some reagent falling within the above definition of vector.Again, the distinction is whether the vaccine includes the alreadysynthesized antigen, or a nucleic acid capable of causing an APC toexpress the antigen in vivo. This strategy constitutes a furtherembodiment of the invention. For rhetorical clarity we have discussedthese approaches as if they were mutually exclusive, but in factcombinations are possible, e.g., a virus vector as above that alsoincorporates a target epitope into a capsid or envelope protein.

Similarly, antigen presenting cells can also be manipulated in vitro andthen themselves used as the active component of a vaccine. Antigenexpression can be conferred by delivering nucleic acid encoded antigenusing any of the transduction techniques known in the art, includingwithout limitation electroporation, viral or bacterial transduction,lipid-mediated transduction, and biolistic bombardment. Alternativelythe APCs may simply be pulsed with antigen. As with any of the otherembodiments of this invention an antigen can be an approximately 8-10amino acid peptide representing a single epitope, a complete protein, apolypeptide encompassing one or more epitopes (including epitopesoriginally derived from multiple proteins) or other forms of antigendescribed above.

Antigens may be used alone or may be delivered in combination with otherantigens or with other compounds such as cytokines that are known toenhance immune stimulation of CTL responses, such as, GM-CSF, IL-12,IL-2, TNF, IFN, IL-18, IL-3, IL-4, IL-8, IL-9, IL-13, IL-10, IL-14,IL-15, G-SCF, IFN alpha, IFN beta, IFN gamma, TGF alpha, TGF beta, andthe like. The cytokines are known in the art and are readily availablein the literature or commercially. Many animal and human tumors havebeen shown to produce cytokines such as IL-4, IL-10, TGF-B that arepotent modulators of the immune response and that protect tumors fromimmune-mediated destruction. The production of IL-4, IL-10 or TGF-B bythe tumors may achieve this protective effect by suppressing theinduction of cellular immunity, including the elaboration of CTLresponses. Alternatively, cytokines that support CTL responses can beexogenously added to help in the balance between induction of anti-tumorcell mediated and non-tumor-destructive humoral responses. Several suchexogenous cytokines show utility in experimental mouse vaccinationmodels which are known to enhance CTL responses, including GM-CSF, IFNand IL-2. An effective exogenous cytokine that may be used is GM-CSF.GM-CSF is reported to enhance the expression of the so called“co-stimulatory” molecules, such as B7-1 or B7-2 on antigen presentingcells (APC), which are important players in the symphony of interactionsthat occur during stimulation of CTL by APC. Moreover, GM-CSF is knownto induce activation of APC and to facilitate growth and differentiationof APC, thereby making these important CTL stimulating cells availableboth in greater numbers and potency.

Delivery of the Antigen

This invention is based in part on the observation that a CTL responseis not sustained using standard vaccine techniques. While not wanting tobe bound by any particular theory, it is thought that T cells do nothave a functional memory that is long-lived. Antibody-mediated B-cellmemory, on the other hand, appears to have a long-lived effector memory.Thus, delivering an antigen that produces a CTL response must be doneover time to keep the patient's immune system appropriately stimulatedto attack the target cells. While it has been suggested that antigensand adjuvants can be prepared as biodegradable microspheres orliposomes, none of these preparations have thus far provided a CTLresponse that is useful for attacking cancer cells or pathogens on along term basis. The delivery must be sustained over the desired periodof time at a level sufficient to maintain the antigen level to obtainthe desired response and that it must be delivered from a reservoirhaving fluid antigen composition that is introduced so that it reachesthe animal's lymphatic system.

Ultimately antigen must find its way into the lymphatic system in orderto efficiently stimulate CTL. However, delivery of antigen according tothe invention can involve infusion into various compartments of thebody, including but not limited to subcutaneous, intravenous,intraperitoneal and intralymphatic, the latter being preferred. Each ofthese various points of infusion results in antigen uptake into thelymphatic system. The relative amounts of antigen needed to induce abeneficial CTL response varies according to the different sites ofinfusion. In general, direct infusion of antigen into the lymph systemis deemed to be the most efficient means of inducing a CTL response, butthat the material difference between the various routes is notnecessarily relevant in terms of the quantity of antigen needed, or, interms of the operating parameters of the invention. The pump systems ofthe invention are capable of delivering material quantities of antigenin a range that makes the invention suitable for inducing CTL responsethrough delivery to all compartments of the body. CTL stimulation basedon delivery of antigen via various routes will be variable, based on theproperties of different antigens, which will reflect factors thatinfluence antigen behavior in the body and its rate of equilibration to(or longevity in) the lymph, such an antigen stability in the bodyfluid, solubility of antigen in body fluid, binding affinity for HLA andpotency as a stimulator of CTL.

It is most efficient, and therefore, preferred, that the introduction isdone as directly as possible to the lymphatic system to avoid thedestruction of the antigen by metabolism in the body. When introductionof a fluid antigen composition occurs subcutaneously, larger quantitiesof antigen are needed to assure enough antigen reaches the lymphaticsystem. Such subcutaneous injection is contemplated by this invention ifit can be justified by factors such as cost, stability of the antigen,how quickly the antigen gets to the lymph system, how well itequilibrates with the lymph, and other factors that the attending doctoror specialist will recognize. Subcutaneous delivery will generallyrequire 100 to 1000 times more antigen than direct delivery to the lymphsystem. It is preferable, therefore, that the antigen composition isintroduced through a device for local administration to the lymphaticsystem, e.g. the spleen, a lymph node, or a lymph vessel. The device forlocal administration may be positioned outside the patient or implantedinto the patient. In either case, the device will have a reservoir tohold the fluid antigen-containing composition, a pump to transfer thecomposition, and a transmission channel leading from the reservoir to bedirected to the preferred region of administration in the patient'sbody. In either case it is preferably portable.

For the device positioned outside the patient's body (the externaldevice), there are numerous devices used for delivering insulin todiabetic patients that are useful in this invention. Generally these arecomprised of a reservoir for holding the antigen composition (instead ofinsulin), a programmable pump to pump the composition out of thereservoir, a transmission channel or line for transmitting thecomposition, and a means to introduce the composition into the animal'sbody to ultimately reach the lymphatic system.

The pump employed may be a roller/peristaltic pump, a syringe pump, apiston/valve pump, a gas pressure pump, or the like that has a powersource (generally a battery for portability) that is programmable todeliver the desired level of antigen composition to the patient's bodyand the lymphatic system. A further discussion of the operation of thesepumps may be found “Insulin Pump Therapy” by E. Austenst and T. Stahl,Walter de Gruyter, Berlin, N.Y. (1990), at Chapter 3. A list of pumpsavailable at that time that are useful for this invention are given inTable IV.

More recent versions of these pumps are available from the manufacturersshown.

TABLE IV Manufacturer/ Weight Name distributor (g) Size (mm) NordiskInfusor Nordisk 180 100 × 60 × 20  Betatron I CPI/Lilly 197 99 × 66 × 20RW 90 P/RW 91 Dahedi/EA Satorius 110 109 × 42 × 22  P/RW 92 InstrumentsMRS 4-Infuser Disetronic 100 75 × 53 × 19 B-D 1000 Becton-Dickinson 1317  8 × 57 × 20 Nordisk Infusor Nordisk 180 113 × 65 × 22  MK 11 MRS3-Infuser Disetronic 100 75 × 53 × 18 A S8 MP Autosyringe/Travenol 161102 × 64 × 19  Betatron 11 CPULilly 197 99 × 66 × 20 Minimed 504Pacesetter/Haselmeyer 106 86 × 21 × 51 Minimed 404 S* Pacesetter 106 86× 21 × 51 MRS I/H-Tron Disetronic/Hoechst 100 75 × 53 × 18 *not yetcommercially available

Particularly useful pumps are the Disetronic H-Tron V 100 Insulin Pumpfrom Disetronic Medical Systems, Burgdorf, Switzerland and the Minimed507 Insulin Pump from MiniMed Inc., 12744 San Fernando Road, Sylmar,Calif. 91342. The MiniMed is particularly useful in that it allowsprogramming a bolus without looking at the pump through a series ofaudio tones (settable in either 0.5 or 1.0 unit increments) and allowsprogramming a bolus for delivery over an extended period of time—from 30minutes to 4 hours. It provides up to 12 basal rates (or profiles) thatcan be programmed per 24 hours from 0.0-25 units/hour in 0.1 unitincrements. The device allows for the temporary increase or decrease ofa set basal rate from 30 minutes to 24 hours in 30 minute increments,Other features relating to safety, time display, memory, etc. areavailable from the manufacturer.

The reservoir for the antigen composition should be large enough fordelivery of the desired amount of antigen over time and is easilyrefillable or replaceable without requiring the user to reinsert themeans for introducing the antigen composition to the lymph system.

In preparing the antigen compositions of this invention, a composition(preferably aqueous) is prepared to be compatible with the lymph systemand is physiologically acceptable to the animal being treated. Inpreparing the antigen compositions useful in this invention oneconsiders the physicochemical properties of the antigen such as theisoelectric point, molecular weight, glycosylation or otherpost-translational modification, and overall amino acid composition.These properties along with any known behavior of the drug in differentsolutions (e.g. different buffers, cofactors, etc.) as well as its invivo behavior will help guide the choice of formulation components. Oneparameter that impacts all the major degradation pathways is thesolution pH. Thus, the initial formulations also assess the pHdependence of the degradation reactions and the mechanism fordegradation can often be determined from the pH dependence to determinethe stability of the protein in each solution. Rapid screening methodsusually involve the use of accelerated stability at elevatedtemperatures (e.g. 40° C.) using techniques known in the art.

In general the antigen compositions useful in this invention will beprepared suitable for parenteral injection, in very small quantities. Assuch a composition must be free of contamination and have a pHcompatible with the lymph system. However, because very small quantitiesof the antigenic composition will be delivered it need not be the samepH as blood or lymph, and it need not be aqueous-based. For antigensthat are less soluble a suitable cosolvent or surfactant may be used,such as dimethyl sulfoxide (DMSO) or PLURONIC brand surfactants. The pHrange that is compatible is from about 6.7-7.3 and can be prepared usingwater for injection to meet USP specifications (see Remington: TheScience and Practice of Pharmacy, Nineteenth Edition; Chapters 86-88).Generally, a standard saline solution that is buffered with aphysiologically acceptable weak acid and its base conjugate, e.g., aphosphate or citrate buffering system, will be the basis of the antigencomposition. In some cases, a small amount of an antioxidant may beuseful to stabilize the composition and prevent oxidation. Factors toconsider in preparing the antigen compositions may be found in the 1994American Chemical Society book entitled “Formulation and Delivery ofProteins and Peptides” (Acs Symposium Series, No. 567) by Jeffery L.Cleland and Robert Langer (Editor)).

For nucleic acid encoded antigens similar considerations apply, althoughthe variety of physico-chemical properties encountered with polypeptidesis absent, so that acceptable formulations will have nearly universalapplicability. As seen in examples 6-10, plasmid DNA in standardphosphate buffered saline (PBS) is an acceptable and effectiveformulation. In some embodiments of the invention, DNA is administeredcontinuously or intermittently at short intervals, from a reservoir wornon, or implanted in, the patient's body. It is preferable that the DNAbe maintained in a soluble, stable form at or near body temperature overa period of time measured minimally in days. In such applications wherethe formulated nucleic acid will be delivered from a reservoir over aperiod several days or longer, the stability of the nucleic acid at roomor body temperature for that period of time, as well as its continuedsterility, take on increased importance. The addition of bacteriostaticagents (e.g. benzyl or ethyl alcohol) and chelating agents (e.g. EDTA)is useful toward these ends. Formulations containing about 1-10% ethylalcohol, 0-1% benzyl alcohol, 0.25-0.5 mM EDTA and a citrate-phosphatebuffer of pH 7.4-7.8 generally perform well. Such formulations are alsoappropriate for bolus injections.

Generally the amount of the antigen in the antigen composition will varyfrom patient to patient and from antigen to antigen, depending on suchfactors as the activity of the antigen in inducing a response and theflow rate of the lymph through the patient's system. In general theantigen composition may be delivered at a rate of from about 1 to about500 microliters/hour or about 24 to about 12000 microliters/day. Theconcentration of the antigen is such that about 0.1 micrograms to about10,000 micrograms of the antigen will be delivered during 24 hours. Theflow rate is based on the knowledge that each minute approximately about100 to about 1000 microliters of lymph fluid flows through an adultinguinal lymph node. The objective is to maximize local concentration ofvaccine formulation in the lymph system. A certain amount of empiricalinvestigation on patients will be necessary to determine the mostefficacious level of infusion for a given vaccine preparation in humans.

To introduce the antigen composition into the lymphatic system of thepatient the composition is preferably directed to a lymph vessel, lymphnode, the spleen, or other appropriate portion of the lymph system.Preferably, the composition is directed to a lymph node such as aninguinal or axillary node by inserting a catheter or needle to the nodeand maintaining the catheter or needle throughout the delivery. Suitableneedles or catheters are available made of metal or plastic (e.g.polyurethane, polyvinyl chloride [PVC], TEFLON, polyethylene, and thelike). In inserting the catheter or needle into the inguinal node forexample, the inguinal node is punctured under ultrasonographic controlusing a Vialon™ Insyte-W™ cannula and catheter of 24G3/4 (BectonDickinson, USA) which is fixed using Tegadenn transparent dressingTegaderm™ 1624, 3M, St. Paul, Minn. 55144, USA). This procedure isgenerally done by an experienced radiologist. The location of thecatheter tip inside the inguinal lymph node is confirmed by injection ofa minimal volume of saline, which immediately and visibly increases thesize of the lymph node. The latter procedure allows confirmation thatthe tip is inside the node and can be performed to ensure that the tipdoes not slip out of the lymph node can be repeated on various daysafter implantation of the catheter. In case the tip did in fact slip outof location inside the lymph node, a new catheter can be implanted.

In another embodiment, the antigen is delivered to the lymphatic systemthrough an article of manufacture that is implanted in the animal,preferably at or near a site of a lymphatic organ. The article willinclude a pump that can deliver the antigen at a controlled rate over apre-determined period of time and is suitable for use in the host.Several devices are known in the art for the delivery of agents (such asdrugs) in humans or animals and these can be used or adapted for use inthe present invention.

The implantable device will be similar to the external device discussedabove in that it comprises a reservoir of a physiologically-acceptable,aqueous, antigen-containing composition that is capable of inducing aCTL response in an animal, a pump positioned in association with thereservoir to deliver the composition at a defined rate, a transmissionchannel to discharge the composition from the reservoir, and optionallya delivery line connected to the transmission channel, which deliveryline is of a size suitable for positioning in the animal and fordelivery of the composition in a manner that reaches the lymphaticsystem of the animal.

Preferably the pump in the implantable device is an osmotic pump of thetype used in the ALZET® model device or the DUROS™ model devicepioneered by Alza Corporation, Palo Alto, Calif. or in a device made byPharmetrix and exemplified in U.S. Pat. No. 4,838,862. The osmotic pumputilizes the osmotic effect using a membrane permeable to water butimpermeable to a solute. Osmotic pressure built up in a device is usedto deliver a composition at a controlled rate over time. A review byGiancarlo Santus and Richard Baker of “Osmotic Drug Delivery: A Reviewof the Patent Literature” in the Journal of Controlled Release 35 (1995)1-21, provides useful guidelines for the type of osmotic pumps that areuseful in this invention. The osmotic pump forces the compositionthrough a discharge orifice to discharge the composition, Optionally adelivery line connects to the discharge orifice to position the linesuitably for delivery to the lymphatic system of the animal. Patentsthat describe devices useful in this invention include the followingU.S. patents: (A) U.S. Pat. No. 3,604,417 assigned to American Cyanamid;(B) U.S. Pat. Nos. 4,838,862; 4,898,582; 5,135,498; 5,169,390; and5,257,987 all assigned to Pharmetrix, (C) U.S. Pat. Nos. 4,340,048;4,474,575; 4,552,651; 4,619,652; 4,753,651; 3,732,865; 3,760,804;3,760,805; 3,929,132; 3,995,632; 4,034,756; 4,350,271; 4,455,145;5,017,381; 5,023,088; 5,030,216; 5,034,229; 5,037,420; 5,057,318;5,059,423; 5,110,596; 5,110,597; 5,135,523; 5,137,727; 5,174,999;5,209,746; 5,221,278; 5,223,265; 3,760,984; 3,987,790; 3,995,631;4,203,440; 4,286,067; 4,300,558; 4,304,232; 4,340,054; 4,367,741;4,450,198; 4,855,141; 4,865,598; 4,865,845; 4,872,873; 4,929,233;4,963,141; 4,976,966, all assigned to Alza Corp. Each of the foregoingpatents are incorporated herein by reference.

A basic osmotic pump device incorporates a housing containing a chamberfor storing the antigen containing composition to be delivered,separated from a compartment containing an osmotic salt material by abarrier that is moveable under pressure such as a piston or a flexibleimpermeable membrane. The compartment containing the osmotic salt isseparated from osmotic fluid by a semipermeable membrane. In someembodiments, a fluid barrier, such as a foil sheet, isolates the osmoticsalt chamber from the osmotic fluid, keeping the pump inactivated untilremoval of the barrier immediately prior to use. Other osmotic pumpdevices use body fluid as the osmotic fluid. In these devices asemipermeable membrane separates the osmotic salt compartment from bodyfluids, and the pump is activated once inserted into the body underexposure to body fluids. In either case, volumetric expansion of theosmotic salt compartment drives the expulsion of the stored antigen fromthe compartment and into the surrounding environment of the body. Thesepumps have been highly successful at achieving steady-state pumping anddelivery of agents. The pumps are of a small size that can be insertedinto a patient, with flexible consideration as to location. This isimportant in the case of CTL vaccines, since the inventor has determinedthat efficient induction of CTL responses is contingent on the antigenor antigen expression system being delivered into the lymphatic system,in order to ultimately achieve antigen delivery into a lymphatic organsuch as the spleen. Antigen delivered into a lymph node is 100-1000times more efficient at inducing CTL responses compared withconventional subcutaneous delivery. A modification to the osmotic pumpincorporates a microcatheter attachment (i.e., the optional deliveryline) at its discharge end, such that when the pump is implantedproximal to a lymphatic organ, such as a lymph node, the catheter can beinserted into the organ to facilitate delivery of the vaccine directlyinto the lymphatic system.

Prior to the administration of the antigen using any of the abovevehicles, methods may be used to assist in the determination of theoptimum location for the antigen delivery. For example, when using theosmotic pump, radiography may be used to image a patient's lymphaticflow, to determine where relatively high lymphatic drainage occurs, inorder to decide upon an insertion position for the osmotic pump thatmaximizes delivery into the lymphatic system. Since each patient hasunique lymphatic drainage profiles, imaging would be conducted for eachindividual prior to insertion of osmotic pump for delivery of antigen.When using direct cannulation of the lymphatic vessel, such as in theuse of osmotic or insulin pumps to deliver antigen, ultrasound can beused to position the needle directly into the lymphatic vessel and tomonitor its positioning over the period of treatment.

The following non-limiting examples are illustrative of the presentinvention.

EXAMPLES Materials and Methods For Examples 1-5

Mice: The generation of T cell receptor transgenic mice (TCR+ mice) inwhich approx. 90% of the CD8+ T cells express a TCR recognizing theimmunodominant LCMV-glycoprotein epitope (gp-peptide aa33-41,p33:K.AVYNFATC-SEQ ID NO:569) presented on H-2D^(b), has been describedin detail. All experimental mice were between 8 and 12 weeks of age andbred and held under strict pathogen free conditions at the Institut FürLabortierkunde at the University of Zurich.

Viruses: LCMV (Armstrong strain) was originally obtained from Dr. M. B.A. Oldstone, Scripps Clinics and Research Foundation, LaJolla, SanDiego, Calif. Seed virus was grown on BHK cells and plaqued on MC57cells using an immunological focus assay, as described previously.

Osmotic pump: ALZA model #1007b.

In vivo protection assays for specific CTL activity: The in vivo assayfor the detection of CTL activity by challenge infections with LCMV hasbeen described in detail previously (Oehen et al. 1991). Briefly, miceare intravenously challenged with 2×10³ pfu of LCMV (Armstrong), After 4days the titer of LCMV is determined using the above mentionedimmunological focus assay.

Primary ex vivo cytotoxicity against LCMV-gp: Mice were injectedintravenously with 10 μg of p33. After 36 hours spleen single cellsuspensions were coincubated for 5 h with ⁵¹Cr-labeled syngeneic EL-4(H-2^(b)) target cells, that were either pulsed with p33 or leftunpulsed. Specific lysis was calculated as [(experimental ⁵¹Crrelease−spontaneous ⁵¹Cr release)/(total ⁵¹Cr release−spontaneous ⁵¹Crrelease)×100%].

LCMV induced foot pad swelling reaction: Mice were infected with LCMV(Armstrong) by intradermal injection into the hind footpad (5000 pfu in30:1), Footpad thickness was measured daily with a spring loadedcaliper. Footpad swelling is calculated as (measured thickness−thicknessbefore injection)/(thickness before injection).

Example 1

Continuous Release of Peptide Antigen Using Osmotic Pump Induces PotentCTL Response in C5BL/6 Mice

C57BL/6 mice were either intravenously injected with a single dose of 50μg p33 (including 500 ng GM-CSF) (circles) or were implanted with amicro-osmotic pump releasing a mixture of 50 μg of p33 and 500 ng GM-CSFover a time period of 7 days (triangles), or were left naïve (data notshown). After 7 days mice were sacrificed to prepare single cellsuspensions from the spleen. Spleen cells were restimulated in vitro for5 days by p33 pulsed in the presence of low amounts of IL-2. Specificcytotoxicity was measured using ⁵¹Cr-labeled EL-4 target cells pulsedwith p33. Specific lysis of EL-4 target cells without p33 was less than16% for all effectors. The results are shown in FIG. 1.

Example 2

Continuous Release of Antigen Induces CTL Immunity Against Virus inC57BL/6 Mice

C57BL/6 mice were either intravenously injected with a single dose of 50μg p33 (including 500 ng GM-CSF. Pharmingen) or were implanted with amicrosomotic pump releasing a mixture of 50 μg of p33 and 500 ng GM-CSFover a time period of 7 days, or were left naive. After 7 days specificCTL activity was assessed in vivo using anti-viral protection assays.C57BL/6 mice were intravenously challenged with LCMV Armstrong strain(2×10³ p.f.u.). After 4 days mice were sacrificed and LCMV titers weredetermined in spleens using an immunological focus assay. Mice implantedwith osmotic pump showed significantly lower virus titers indicatingactive CTL immunity against the virus (Table V).

TABLE V C57BL/6 Mice Virus Titer (loglo) Single injection 4.2 Singleinjection 4.6 Single injection 4.0 Pump delivered 2.2 Pump delivered 1.8Pump delivered 2.0 Unprimed 4.8 Unprimed 3.8 Unprimed 4.4

Example 3

Continuous Release of Antigen Maintains Potent CTL Effectors in TCRTransgenic Mice

TCR transgenic mice were either intravenously injected with a singledose of 50 μg p33 (circles) or were implanted with a microsomotic pumpreleasing a mixture of 50 μg of p33 (triangles), or left naïve(squares). After 36 hours mice were sacrificed to prepare single cellsuspensions from the spleen which were assayed ex vivo for p33-specificcytotoxicity using ⁵¹Cr-labeled EL-4 target cells pulsed with p33.Similarly mice were either intravenously injected with a single dose of50 μg p33 (circles) or were implanted with a micro-osmotic pumpreleasing a mixture of 50 μg of p33 over a time period of 7 days(triangles), or were left naïve (squares). After 7 days mice weresacrificed to prepare single cell suspensions from the spleen to assayex vivo p33-specific cytotoxicity using ⁵¹Cr-labeled EL-4 target cellspulsed with p33. Specific lysis of EL-4 target cells without p33 wasless than 18% for all effectors. The results are shown in FIGS. 2A and2B.

Continuous Release of Antigen Maintains Protective CTL Response AgainstVirus Infection

After 7 days TCR transgenic mice were challenged by intradermal LCMVinjection into their hind foot pads (2×10³ pfu in 30 μl). The absence ofa foot pad swelling reaction, as observed in mice with an implanted pump(triangles), indicates that at the time point of injection there wasactive CTL immunity inhibiting local replication of the virus in thefoot pad. In contrast, foot pad swelling, as observed in mice injectedwith the peptide as a single bolus (circles) and naive control mice(data not shown), indicated that LCMV successfully replicated in thefoot pad in the absence of protective CTL. The results are shown in FIG.2C.

Example 4

Direct Delivery of Antigen into Lymphatic Organ Dramatically IncreasesEfficiency of CTL Induction

TCR transgenic mice were injected with graded doses of gp-peptide p33either subcutaneously (S.C.), intravenously (I.V.) or directly into thespleen (I.S.) via a small abdominal incision. The efficiency of CTLinduction was assessed by measuring gp-specific CTL activity 24 hoursafter injection. CTL activity is known to peak one day after injectionof peptide. Mice were sacrificed to prepare single cell suspensions fromdraining lymph nodes or from spleen to assay ex vivo p33-specificcytotoxicity using ⁵¹Cr-labeled EL-4 target cells pulsed with p33.Specific lysis of EL-4 target cells without p33 was less than 12% forall effectors. The results are shown in FIG. 3.

Example 5

Dendritic Cells Purified from Mice Receiving Intrasplenic Injection ofPeptide Potently Stimulate CTL

The effect of directing peptide delivery into lymphatic system wasassessed. Peptide p33 was injected either i.v., s.c. or directly intothe spleen of wild-type C57BL/6 mice. After 2 hours, DCs were isolatedfrom the spleen of animals injected either i.s. or i.v., andadditionally from the regional draining lymph nodes of animals injecteds.c. Cells isolated from these tissues were sorted for DCs usingmagnetic beads coupled with a monoclonal antibody recognizing theintegrin-alpha chain, a marker specific for DCs in spleen and lymphnodes. The positively and the negatively sorted cell fractions werecompared regarding their capacity to in vitro stimulate naive CD8+ Tcells from TCR transgenic mice specific for LCMV-gp. Only when peptidehad been directly injected into the spleen, the DC containing cellfraction stimulated CTL to proliferate, as measured by ³H-thymidineuptake. This indicated that CTL induction after direct injection ofpeptide into lymphatic organs reflected efficient loading of DCs withpeptide. In contrast, the fraction depleted for DC did not induceproliferation and DCs isolated from lymphoid organs of i.v. and s.cinjected mice were not effective stimulators. The results are shown inFIG. 4.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

Example 6

Induction of CTL Response with Naked DNA is Most Efficient byIntra-Lymph Node Immunization.

In order to quantitatively compare the CD8⁺ CTL responses induced bydifferent routes of immunization we used a plasmid DNA vaccine(pEGFPL33A) containing a well-characterized immunodominant CTL epitopefrom the LCMV-glycoprotein (G) (gp33; amino acids 33-41) (Oehen, S., etal. Immunology 99, 163-169 2000), as this system allows a comprehensiveassessment of antiviral CTL responses. Groups of 2 C57BL/6 mice wereimmunized once with titrated doses (200-0.02 μg) of pEGFPL33A DNA or ofcontrol plasmid pEGFP-N3, administered i.m. (intramuscular), i.d.(intradermal), i.spl. (intrasplenic), or i.ln. (intra-lymph node).Positive control mice received 500 pfu LCMV i.v. (intravenous). Ten daysafter immunization spleen cells were isolated and gp33-specific CTLactivity was determined after secondary in vitro restimulation. As shownin FIG. 6, i.m. or i.d. immunization induced weakly detectable CTLresponses only when high doses of pEFGPL33A DNA (200 μg) wereadministered. In contrast, potent gp33-specific CTL responses wereelicited by immunization with only 2 μg pEFGPL33A DNA i.spl. and with aslittle as 0.2 μg pEFGPL33A DNA given i.ln. (FIG. 6; symbols representindividual mice and one of three similar experiments is shown).Immunization with the control pEGFP-N3 DNA did not elicit any detectablegp33-specific CTL responses (data not shown).

Example 7

Intra-Lymph Node Immunization is the Most Efficient Way to InduceAntiviral Anamnestic CTL Responses.

Similar thresholds for CTL detection were observed when a differentreadout system was used. Groups of 2 C57BL/6 mice were immunized oncewith titrated doses of pEFGPL33A DNA (0.2-200 μg) and positive controlmice received 500 pfu LCMV i.v., as above. Ten days later they werechallenged with 5×10⁴ pfu LCMV i.v. Four days after challenge spleencells were isolated and ex vivo CTL activity was assayed. This timepoint is too early to detect any primary CTL response to LCMV infectionin naive mice (FIG. 7, Controls), but it allows the detection ofanamnestic CTL responses in mice which have been previously immunized(FIG. 7, LCMV). As before, mice immunized with 200 μg intramuscularlyshowed only weak anamnestic CTL responses following LCMV challenge,which were not detectable when lower immunizing doses of DNA were used(FIG. 7). Those immunized by the i.spl. route showed strong anamnesticCTL responses which titered out at an immunizing dose of 2 μg pEFGPL33ADNA, while the i.ln. route of immunization was again more efficient withanamnestic CTL responses detectable when only 0.2 μg pEFGPL33A DNA wasadministered (FIG. 7).

These results from examples 6 and 7 clearly demonstrate thatadministration of plasmid DNA directly into lymphoid tissues is 100- to1000-fold more efficient than intradermal or intramuscular routes forthe induction of CTL responses. In addition, they show that theintra-lymph node route is around 10-fold more efficient than theintrasplenic route.

Example 8

Naked DNA Elicits Superior Protection Against Systemic and PeripheralVirus Infection by Intra-Lymph Node Compared to IntramuscularImmunization.

To examine whether the enhanced CTL responses elicited following i.ln.immunization with plasmid DNA were able to qualitatively influenceantiviral immunity, we used challenge infections with LCMV or withrecombinant vaccinia virus expressing the LCMV-G (Vacc-G2) as models ofsystemic and peripheral virus infection, respectively. When systemicantiviral immunity was assessed by challenging the immunized mice(groups of 3 C57BL/6 mice) with a high dose of LCMV i.v. (500 pfu), micewhich had been immunized once with 200 μg pEGFPL33A DNA i.m. showed onlypartial and incomplete protection against systemic LCMV challenge, whilethose which had received 20 μg of pEFGPL33A DNA by the i.spl. or i.ln.routes were completely protected (FIG. 8A).

Eradication of Vacc-G2 infection from peripheral organs such as ovaries,is dependent upon the presence of high levels of recently activatedeffector CD8⁺ T cells (Kündig, T. M. et al. Proc. Natl. Acad. Sci. USA93, 9716-9723, 1996; Bachmann, M. F., et al. Proc. Natl. Acad. Sci. USA94, 640-645, 1997). Groups of 3 C57BL/6 mice were immunized four timesat 6 day intervals with pEFGPL33A DNA administered either i.m. (100 μgper immunization) or i.ln. (10 μg per immunization). Five days after thelast immunization they were challenged with 5×10⁶ pfu Vacc-G2 i.p. andvaccinia titers in ovaries were assessed after a further 5 days.Repeated i.m. immunization with pEFGPL33A DNA had no influence on thegrowth of Vacc-G2 in peripheral tissues (FIG. 8B). In contrast, micewhich were repetitively immunized with pEFGPL33A DNA by the i.ln. routewere completely protected against peripheral infection with Vacc-G2(FIG. 8B).

These results illustrate that although repeated i.m. immunization withnaked DNA induced detectable CTL responses, these were never ofsufficient magnitude to offer protection against virus infection. Incontrast, immunization with 10-fold lower amounts of DNA directly intolymphoid organs elicited quantitatively and qualitatively stronger CTLresponses, which gave complete protection against systemic or peripheralvirus challenge.

Example 9

Intra-Lymph Node DNA Immunization Elicits Anti-Tumor Immunity

To examine whether the potent CTL responses elicited following i.ln.immunization were able to confer protection against peripheral tumors,groups of 6 C57BL/6mice were immunized three times at 6-day intervalswith 10 μg of pEFGPL33A DNA or control pEGFP-N3 DNA. Five days after thelast immunization small pieces of solid tumors expressing the gp33epitope (EL4-33) were transplanted s.c. into both flanks and tumorgrowth was measured every 3-4 d. Although the EL4-33 tumors grew well inmice that had been repetitively immunized with control pEGFP-N3 DNA(FIG. 9), mice which were immunized with pEFGPL33A DNA i.ln. rapidlyeradicated the peripheral EL4-33 tumors (FIG. 9).

Example 10

Differences in Lymph Node DNA Content Mirrors Differences in CTLResponse Following Intra-Lymph Node and Intramuscular Injection.

pEFGPL33A DNA was injected i.ln. or i.m. and plasmid content of theinjected or draining lymph node was assessed by real time PCR after 6,12, 24, 48 hours, and 4 and 30 days. At 6, 12, and 24 hours the plasmidDNA content of the injected lymph nodes was approximately three ordersof magnitude greater than that of the draining lymph nodes followingi.m. injection. No plasmid DNA was detectable in the draining lymph nodeat subsequent time points (FIG. 10). This is consonant with the threeorders of magnitude greater dose needed using i.m. as compared to i.ln.injections to achieve a similar levels of CTL activity. CD8^(−/−)knockout mice, which do not develop a CTL response to this epitope, werealso injected i.ln. showing clearance of DNA from the lymph node is notdue to CD8+ CTL killing of calls in the lymph node. This observationalso supports the conclusion that i.ln. administration will not provokeimmunopathological damage to the lymph node.

Example 11

Stability of Plasmid in Different Formulations

DNA is a relatively stable molecule in the kind of formulations ofinterest to test and thus little loss of material would be noted if thetotal amount of DNA were to be measured. Instead, the ratio ofsupercoiled to open-circle DNA was measured. Since a single nickanywhere in either strand of the DNA molecule will allow a supercoiledplasmid to relax to an open circle conformation this is an exquisitelysensitive indication of damage to the DNA backbone. Plasmid wasformulated, placed in vials in triplicate and incubated at 37° C. After1, 3 and 7 days aliquots were removed, subjected to anion exchange HPLC,and the peak areas corresponding to supercoiled and open-circle DNAcompared (see FIG. 11). Nine formulations were tested:

10% Ethanol, 0.25 mM EDTA, Citrate Phosphate pH 7.6

10% Ethanol, 0.25 mM EDTA, Citrate Phosphate pH 7.4

1% Ethanol, 0.5 mM EDTA, Citrate Phosphate pH 7.4

1% Ethanol, 0.5 mM EDTA, 1×PBS pH 7.4

0.5% Benzyl Alcohol, 0.25 mM EDTA, Citrate Phosphate pH 7.6

1% Benzyl Alcohol, 1% Ethanol, 0.5 mM EDTA, Citrate Phosphate pH 7.6

1% Benzyl Alcohol, 1% Ethanol, 0.5 mM EDTA, 0.1M TRIS pH 7.4

1% Benzyl Alcohol, 1% Ethanol, 0.5 mM EDTA, 0.1M TRIS pH 8.2

1% Benzyl Alcohol, 1×PBS pH 8.2

Citrate Phosphate Buffer pH. 7.4 was made by mixing 9.15 parts (byvolume) of 0.1M citric acid with 90.85 parts of 0.2M Sodium PhosphateDibasic. Citrate Phosphate Buffer pH. 7.6 was made by mixing 6.35 parts(by volume) of 0.1M citric acid with 93.65 parts of 0.2M SodiumPhosphate Dibasic. These solutions were then added to the othercomponents to create a 2× buffer which was mixed with a equal volume ofDNA in water. Thus the final concentrations of citrate and phosphate inthe above buffers was on the order of 3 mM and 90 mM, respectively.

Formulations 1-3 and 6 gave superior results (see FIG. 11).

Example 12

Stability of Formulated Plasmid in Operating MINIMED 407C InfusionPumps.

Using a modification (final concentrations of 0.1 M sodium phosphatedibasic and 0.05 M citric acid; pH 7.6±0.2) of formulation 6 above,aliquots of 80, 160, and 320 μg DNA/ml were prepared and loaded intriplicate into MINIMED 3.0 reservoir syringes. A 200 μl sample wasdispensed and the reservoir syringes were inserted into MINIMED 407Cinfusion pumps and assembled with SILHOUETTE infusion sets fitted with3.1 mm catheters. The pump assemblies, set to dispense 10 μl/hour andwith the catheters inserted into collection vials, were placed in 37° C.incubators. At 4 and 8 days the catheters were briefly detached and 200μl bolus samples dispensed directly from the reservoir. Theconcentration of supercoiled DNA was determined for each sample by anionexchange HPLC and the use of a standard curve constructed with knownconcentrations of DNA. Plotting the resultant concentrations versus timeallows one to derive a slope indicating the rate of loss of supercoiledDNA. The average (of the triplicate samples) rates of loss were−0.056±1.88, 0.24±1.01, and 0.048±0.49 μg DNA/day for the 80, 160, and320 μg DNA/ml samples, respectively. None of these differ significantlyfrom zero.

Example 13

Administration of a DNA Plasmid Formulation of a Therapeutic Vaccine forMelanoma to Humans.

SYNCHROTOPE TA2M, a melanoma vaccine encoding HLA-A2-restrictedtyrosinase epitopes was formulated in 1% Benzyl alcohol, 1% ethylalcohol, 0.5 mM EDTA, citrate-phosphate, pH 7.6. Aliquots of 80, 160,and 320 μg DNA/ml were prepared for loading into MINIMED 407C infusionpumps. The catheter of a SILHOUETTE infusion set is placed into aninguinal lymph node visualized by ultrasound imaging. The assembly ofpump and infusion set was originally designed for the delivery ofinsulin to diabetics and the usual 17 mm catheter has been substitutedwith a 31 mm catheter for this application. The infusion set is keptpatent for 4 days (approximately 96 hours) with an infusion rate ofabout 25 μl/hour resulting in a total infused volume of approximately2.4 ml. Thus the total administered dose per infusion will beapproximately 200, 400, and 800 μg, respectively, for the threeconcentrations described above. Following an infusion subjects will begiven a 10 day rest period before starting a subsequent infusion. Giventhe continued residency of plasmid DNA in the lymph node afteradministration (as in example 10) and the usual kinetics of CTL responsefollowing disappearance of antigen, this schedule will be sufficient tomaintain the immunologic CTL response.

1. A method of inducing a CTL response in a mammal, which methodcomprises: delivering a liquid comprising an antigen directly to an areaof high lymphatic drainage or a lymphatic system of the mammal at alevel sufficient to induce an antigen-specific CTL response in themammal; and maintaining the antigen in the mammal's lymphatic systemover time sufficient to induce the CTL response.
 2. The method of claim1, wherein the CTL response induced is an increase in a preexisting CTLresponse.
 3. The method of claim 1, further comprising obtaining asustained CTL response in the mammal.
 4. The method of claim 3, furthercomprising detecting the sustained antigen-specific effector CTLresponse in the mammal, wherein the detecting comprises an assayselected from the group consisting of a cytokine assay, a chromiumrelease assay, an antiviral protection assay, virus titer, animmunofluorescence assay, a tumor growth inhibition assay, tumor sizereduction, a CTL assay, inhibition of tumor metastasis, increase in lifeexpectancy, infectious disease recovery, inflammatory reaction assay,and observation of the health of the mammal.
 5. The method of claim 1,wherein delivering the liquid comprising the antigen comprises deliveryto an inguinal or axillary lymph node.
 6. The method of claim 1, whereindelivering the liquid comprising the antigen comprises delivery to anarea of high lymphatic drainage in the mammal.
 7. The method of claim 1,wherein delivering the liquid comprising the antigen comprises deliveryin a single bolus.
 8. The method of claim 1, wherein delivering theliquid comprising the antigen comprises delivery in a sustained,repeated, or continuous manner.
 9. The method of claim 1, whereindelivering the liquid comprising the antigen comprises delivery from anexternal or implantable, sustained-release pump.
 10. The method of claim1, wherein the antigen is a disease-matched antigen.
 11. The method ofclaim 1, wherein the antigen is a patient-matched antigen.
 12. Themethod of claim 1, wherein the antigen is a non-peptide antigen.
 13. Themethod of claim 1, wherein the antigen is selected from a peptide, apolypeptide, a polypeptide sequence, and a protein.
 14. The method ofclaim 13, wherein the polypeptide consists of 8-10 amino acids.
 15. Themethod of claim 13, wherein the antigen is a polypeptide derived from atumor-associated antigen.
 16. The method of claim 1, wherein the liquidfurther comprises an immunopotentiator.